Methods of treatment utiliziing binding proteins of the interleukin-21 receptor

ABSTRACT

The present invention provides binding proteins and antigen-binding fragments thereof, including human antibodies, that specifically bind to the human interleukin-21 receptor (IL-21R), and methods of using them. The binding proteins can act as, e.g., antagonists of IL-21R activity, thereby modulating immune responses in general, and those mediated by IL-21R in particular. The disclosed compositions and methods may be used, e.g., in diagnosing, treating, and/or preventing IL-21R-associated disorders, e.g., inflammatory disorders, autoimmune diseases, allergies, transplant rejection, and other immune system disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/472,237, filed May 26, 2009, now U.S. Pat. No. 8,178,097, issuedMay 15, 2012, which claims the benefit of priority from U.S. ProvisionalPatent Application No. 61/055,543, filed May 23, 2008, and U.S.Provisional Patent Application No. 61/099,476, filed Sep. 23, 2008; thecontents of these several patent applications are hereby incorporated byreference herein in their entireties.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing filed on May 14, 2012, created/modified on May 11,2012, named 019970690011ST25.txt, having a size in bytes of 324 kB, ishereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to binding proteins and antigen-bindingfragments thereof that bind interleukin-21 receptor (IL-21R), inparticular, human IL-21R, and their use in regulating IL-21R-associatedactivities, e.g., IL-21 effects on the levels of expression of IL-21responsive genes. The binding proteins disclosed herein are useful intreating and/or diagnosing IL-21R-associated disorders, e.g.,inflammatory disorders, autoimmune diseases, allergies, transplantrejection, hyperproliferative disorders of the blood, and other immunesystem disorders. The invention further provides methods for determiningpharmacodynamic and pharmacokinetic properties of the antibodies of theinvention.

2. Related Background Art

Antigens initiate immune responses and activate the two largestpopulations of lymphocytes: T cells and B cells. After encounteringantigen, T cells proliferate and differentiate into effector cells,while B cells proliferate and differentiate into antibody-secretingplasma cells. These effector cells secrete and/or respond to cytokines,which are small proteins (less than about 30 kDa) secreted bylymphocytes and other cell types.

Human IL-21 is a cytokine that shows sequence homology to IL-2, IL-4 andIL-15 (Parrish-Novak et al. (2000) Nature 408:57-63). Despite lowsequence homology among interleukin cytokines, cytokines share a commonfold into a “four-helix-bundle” structure that is representative of thefamily. Most cytokines bind either class I or class II cytokinereceptors. Class II cytokine receptors include the receptors for IL-10and the interferons, whereas class I cytokine receptors include thereceptors for IL-2 through IL-7, IL-9, IL-11, IL-12, IL-13, and IL-15,as well as hematopoietic growth factors, leptin, and growth hormone(Cosman (1993) Cytokine 5:95-106).

Human IL-21R is a class I cytokine receptor. The nucleotide and aminoacid sequences encoding human IL-21 and its receptor (IL-21R) aredescribed in International Application Publication Nos. WO 00/053761 andWO 01/085792; Parrish-Novak et al. (2000) supra; and Ozaki et al. (2000)Proc. Natl. Acad. Sci. USA 97:11439-44. IL-21R has the highest sequencehomology to the IL-2 receptor β chain and the IL-4 receptor α chain(Ozaki et al. (2000) supra). Upon ligand binding, IL-21R associates withthe common gamma cytokine receptor chain (γc) that is shared by receptorcomplexes for IL-2, IL-3, IL-4, IL-7, IL-9, IL-13 and IL-15 (Ozaki etal. (2000) supra; Asao et al. (2001) J. Immunol. 167:1-5).

IL-21R is expressed in lymphoid tissues, particularly on T cells, Bcells, natural killer (NK) cells, dendritic cells (DC) and macrophages(Parrish-Novak et al. (2000) supra), which allows these cells to respondto IL-21 (Leonard and Spolski (2005) Nat. Rev. Immunol. 5:688-98). Thewidespread lymphoid distribution of IL-21R indicates that IL-21 plays animportant role in immune regulation. In vitro studies have shown thatIL-21 significantly modulates the function of B cells, CD4⁺ and CD8⁺ Tcells, and NK cells (Parrish-Novak et al. (2000) supra; Kasaian et al.(2002) Immunity 16:559-69). Recent evidence suggests that IL-21-mediatedsignaling can have antitumor activity (Sivakumar et al. (2004)Immunology 112:177-82), and that IL-21 can prevent antigen-inducedasthma in mice (Shang et al. (2006) Cell. Immunol. 241:66-74).

In autoimmunity, disruption of the IL-21 gene and injection ofrecombinant IL-21 have been shown to modulate the progression ofexperimental autoimmune myasthenia gravis (EAMG) and experimentalautoimmune encephalomyelitis (EAE), respectively (King et al. (2004)Cell 117:265-77; Ozaki et al. (2004) J. Immunol. 173:5361-71; Vollmer etal. (2005) J. Immunol. 174:2696-2701; Liu et al. (2006) J. Immunol.176:5247-54). In these experimental systems, it has been suggested thatthe manipulation of IL-21-mediated signaling directly altered thefunction of CD8⁺ cells, B cells, T helper cells, and NK cells.

Thus, the present invention provides novel therapeutic agents fortreating, e.g., autoimmune diseases that act by blocking the IL-21signaling pathway, i.e., anti-IL-21R antibodies. In order for atherapeutic agent, such as an anti-IL-21R antibody, to be effective invivo, a minimum serum concentration of the anti-IL-21R antibodynecessary to modulate IL-21 responses should be determined; thus amethod that allows accurate determination of such minimum serumconcentration is required.

SUMMARY OF THE INVENTION

The present invention describes the isolation and characterization ofbinding proteins, for example, human antibodies and fragments thereof,that specifically bind to the human and murine IL-21R. The bindingproteins described herein are derived from antibody 18A5, which isdisclosed in U.S. Pat. No. 7,495,085, the entirety of which is herebyincorporated by reference herein. The binding proteins of the presentinvention have a much greater degree of affinity to human and/or murineIL-21R than does the parental 18A5 antibody.

The invention provides, at least in part, IL-21R binding agents (such asbinding proteins and antigen-binding fragments thereof) that bind toIL-21R, in particular, human IL-21R, with high affinity and specificity.The binding proteins, and antigen-binding fragments thereof, of thepresent invention are also referred to herein as “anti-IL-21R bindingproteins” and “fragments thereof,” respectively. In one embodiment, thebinding protein or fragment thereof reduces, inhibits, or antagonizesIL-21R activity. Such binding proteins can be used to regulate immuneresponses or IL-21R-associated disorders by antagonizing IL-21Ractivity. In other embodiments, the anti-IL-21R binding protein can beused diagnostically, or as a targeting binding protein to deliver atherapeutic or cytotoxic agent to an IL-21R-expressing cell. Thus, theanti-IL-21R binding proteins of the invention are useful in diagnosingand treating IL-21R-associated disorders, e.g., inflammatory disorders,autoimmune diseases, allergies, transplant rejection, hyperproliferativedisorders of the blood, and other immune system disorders, as describedmore fully herein.

Accordingly, in one aspect, the binding proteins of the inventionfeature an isolated binding protein (e.g., an isolated antibody) orantigen-binding fragment thereof that binds to IL-21R, in particular,human IL-21R. In certain embodiments, the anti-IL-21R binding protein(e.g., antibody) can have one or more of the following characteristics:(1) it is a monoclonal or single specificity binding protein; (2) it isa human binding protein; (3) it is an in vitro-generated bindingprotein; (4) it is an in vivo-generated (for example, a transgenic mousesystem) binding protein; (5) it inhibits the binding of IL-21 to IL-21R;(6) it is an IgG1; (7) it binds to human IL-21R with an associationconstant of at least about 10⁵ M⁻¹s⁻¹; (8) it binds to murine IL-21Rwith an association constant of at least about 5×10⁴M⁻¹s⁻¹; (9) it bindsto human IL-21R with a dissociation constant of about 10⁻³s⁻¹ or less;(10) it binds to murine IL-21R with a dissociation constant of about10⁻²s⁻¹ or less; (11) it inhibits human IL-21R-mediated proliferation ofhuman IL-21R-expressing BaF3 cells with an IC₅₀ of about 1.75 nM orless; (12) it inhibits murine IL-21R-mediated proliferation of murineIL-21R-expressing BaF3 cells with an IC₅₀ of about 0.5 nM or less; (13)it inhibits human IL-21R-mediated proliferation of humanIL-21R-expressing TF1 cells with an IC₅₀ of about 14.0 nM or less; (14)it inhibits IL-21-mediated proliferation of human primary B cells withan IC₅₀ of about 1.9 nM or less; (15) it inhibits IL-21-mediatedproliferation of human primary CD4⁺ T cells with an IC₅₀ of about 1.5 nMor less; (16) it inhibits IL-21-mediated proliferation of murine primaryCD4⁺ T cells with an IC₅₀ of about 5.0 nM or less; (17) it has a meantotal body clearance of about 0.1-7.5 ml/hr/kg following, e.g.,intravenous (i.v.) administration to animals, e.g., mammals, e.g.,humans, nonhuman primates, rodents; (18) it has a mean eliminationhalf-life of about 20-700 hr following, e.g., i.v., subcutaneous (s.c.),or intraperitoneal (i.p.) administration to animals, e.g., mammals,e.g., humans, nonhuman primates, rodents; (19) it has a meansteady-state volume of distribution of about 40-1500 ml/kg in animals,e.g., mammals, e.g., humans, nonhuman primates, rodents; (20) it has abioavailability of about 35-100% following, e.g., s.c. administration toanimals, e.g., mammals, e.g., humans, nonhuman primates, rodents; (21)it has a mean dose-normalized AUC of about 200-10,000 μg*hr/ml (per 1mg/kg dosage) following, e.g., i.v., s.c., or i.p. administration toanimals, e.g., mammals, e.g., humans, nonhuman primates, rodents; (22)it has a mean dose-normalized C_(max)(maximum serum concentration) ofabout 0.5-30 μg/ml following, e.g., i.v., s.c., or i.p. administrationto animals, e.g., mammals, e.g., humans, nonhuman primates, rodents; and(23) it modulates expression of IL-21 responsive cytokines or IL-21responsive genes.

Nonlimiting illustrative embodiments of the binding proteins of theinvention (the term “binding proteins” also includes and refers toantigen-binding fragments thereof, as appropriate) are referred toherein as AbA-AbZ, and correlation of these terms with terms used inU.S. Provisional Patent Application No. 61/055,543 is presented in Table2A. Other illustrative embodiments of the binding proteins of thepresent invention, i.e., scFv, are referred to herein as H3-H6, L1-L6,L8-L21, and L23-L25, as detailed in Table 2B.

In one embodiment, the binding proteins of the invention are antibodies.In further embodiments, the antibodies are polyclonal, monoclonal,monospecific, polyspecific, nonspecific, humanized, human, single-chain,chimeric, synthetic, recombinant, hybrid, mutated, grafted, invitro-generated and/or multispecific (e.g., bispecific antibodies formedfrom at least two intact antibodies).

One embodiment of the invention is a method of treating or preventing anIL-21R-associated disorder in a subject, comprising administering to thesubject a binding protein or antigen-binding fragment thereof thatspecifically binds to human IL-21R in an amount sufficient to inhibit orreduce immune cell activity in the subject thereby treating orpreventing the disorder, wherein the binding protein or antigen-bindingfragment thereof comprises at least one amino acid sequence that is atleast about 95% identical to an amino acid sequence(s) selected from thegroup consisting of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 165-168, 171-193, 213-229, 240, 242, 244, 246, and 248.

In embodiments of the invention, the binding protein or antigen-bindingfragment can be, e.g., an antibody, an scFv, a V_(H), a V_(L), and/or aCDR.

Another embodiment of the invention is a method of treating orpreventing an IL-21R-associated disorder in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to human IL-21R in an amountsufficient to inhibit or reduce immune cell activity in the subjectthereby treating or preventing the disorder, wherein the binding proteinor antigen-binding fragment thereof comprises at least one amino acidsequence encoded by a nucleotide sequence that is at least about 95%identical to a nucleotide sequence(s) selected from the group consistingof SEQ ID NOs:13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241,243, 245, and 247.

In one embodiment of the invention, the binding protein orantigen-binding fragment comprises at least one amino acid sequenceselected from the group consisting of SEQ ID NOs:14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 165-168, 171-193, 213-229, 240, 242, 244, 246,and 248.

Another embodiment of the invention is a method of treating orpreventing an IL-21R-associated disorder in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to human IL-21R in an amountsufficient to inhibit or reduce immune cell activity in the subjectthereby treating or preventing the disorder, wherein the binding proteinor antigen-binding fragment thereof comprises at least one amino acidsequence that is at least about 95% identical to an amino acidsequence(s) selected from the group consisting of SEQ ID NOs:6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162-195, 213-229, 240, 242,244, 246, and 248, and wherein, if the binding protein orantigen-binding fragment comprises at least one amino acid sequence thatis at least about 95% identical to the sequence(s) selected from thegroup consisting of SEQ ID NOs:6, 8, 10, 12, 163, 164, 169, 170, 194,and 195, then the binding protein or antigen-binding fragment must alsocomprise at least one amino acid sequence that is at least about 95%identical to the amino acid sequence(s) selected from the groupconsisting of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,165-168, 171-193, 213-229, 240, 242, 244, 246, and 248.

Another embodiment of the invention is a method of treating orpreventing an IL-21R-associated disorder in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to human IL-21R in an amountsufficient to inhibit or reduce immune cell activity in the subjectthereby treating or preventing the disorder, wherein the binding proteinor antigen-binding fragment thereof comprises at least one amino acidsequence encoded by a nucleotide sequence that is at least about 95%identical to a nucleotide sequence(s) selected from the group consistingof SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 239, 241, 243, 245, and 247, and wherein, if the binding protein orantigen-binding fragment comprises at least one amino acid sequenceencoded by a nucleotide sequence that is at least about 95% identical tothe sequence(s) selected from the group consisting of SEQ ID NOs:5, 7,9, and 11, then the binding protein or antigen-binding fragment mustalso comprise at least one amino acid sequence encoded by a nucleotidesequence that is at least about 95% identical to the nucleotidesequence(s) selected from the group consisting of SEQ ID NOs:13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

In one embodiment of the invention, the binding protein orantigen-binding fragment comprises at least one amino acid sequenceselected from the group consisting of SEQ ID NOs:6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162-195, 213-229, 240, 242, 244, 246,and 248, wherein, if the binding protein or antigen-binding fragmentcomprises at least one amino acid sequence that is at least about 95%identical to the sequence(s) selected from the group consisting of SEQID NOs:6, 8, 10, 12, 163, 164, 169, 170, 194, and 195, then the bindingprotein or antigen-binding fragment must also comprise at least oneamino acid sequence that is at least about 95% identical to the aminoacid sequence(s) selected from the group consisting of SEQ ID NOs:14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 165-168, 171-193, 213-229,240, 242, 244, 246, and 248.

A further embodiment of the invention is a method of treating orpreventing an IL-21R-associated disorder in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to IL-21R, wherein the bindingprotein or antigen-binding fragment thereof comprises a light chain anda heavy chain, and wherein the heavy chain comprises at least one aminoacid sequence selected from the group consisting of SEQ ID NOs:14, 16,18, 20, 68, 70, 72, 88, 90, 92, 94, 213, 218, 219, 240, and 242. In oneembodiment, the binding protein or antigen-binding fragment comprises aV_(L) domain and a V_(H) domain, and the V_(H) domain comprises at leastone sequence selected from the group consisting of SEQ ID NOs:14, 16,18, and 20.

Another embodiment of the invention is a method of treating orpreventing an IL-21R-associated disorder in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to IL-21R, wherein the bindingprotein or antigen-binding fragment thereof comprises a light chain anda heavy chain, and wherein the light chain comprises at least one aminoacid sequence selected from the group consisting of SEQ ID NOs:22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 74, 76, 78, 80, 82, 84, 86, 96, 98, 100, 102, 104, 106, 108,214-217, 220-229, 244, 246, and 248. In one embodiment, the bindingprotein or antigen-binding fragment comprises a V_(L) domain and a V_(H)domain, and the V_(L) domain comprises at least one sequence selectedfrom the group consisting of SEQ ID NOs:22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 215, 217,221, 223, 225, 227, and 229.

In another embodiment of the methods of the invention, the heavy chaincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:88, 90, 92, 94, 213, 218, 219, 240, and 242, and the lightchain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:96, 98, 100, 102, 104, 106, 108, 214, 216, 220,222, 224, 226, 228, 244, 246, and 248.

One embodiment of the invention is a method of treating or preventing anIL-21R-associated disorder in a subject, comprising administering to thesubject a binding protein or antigen-binding fragment thereof thatspecifically binds to a human IL-21R epitope that is recognized by abinding protein selected from the group consisting of AbA-AbW, H3-H6,L1-L6, L8-L21, and L23-L25, wherein the binding protein orantigen-binding fragment competitively inhibits the binding of a bindingprotein selected from the group consisting of AbA-AbW, H3-H6, L1-L6,L8-L21, and L23-L25 to human IL-21R, in an amount sufficient to inhibitor reduce immune cell activity in the subject thereby treating orpreventing the disorder. In another embodiment, the binding protein orantigen-binding fragment thereof comprises a heavy chain, a light chain,or an F_(v) fragment comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 165-168, 171-193, 213-229, 240, 242, 244, 246, and 248. In yetanother embodiment, the binding protein or antigen-binding fragmentthereof comprises a heavy chain, a light chain, or an F_(v) fragmentcomprising an amino acid sequence encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NOs:13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 239, 241, 243, 245, and 247. In still anotherembodiment, the binding protein specifically binds to an IL-21R epitopethat is recognized by AbO, AbP, AbQ, AbR, AbS, AbT, AbU, AbV, and/orAbW, and the binding protein competitively inhibits the binding of AbO,AbP, AbQ, AbR, AbS, AbT, AbU, AbV, and/or AbW to human IL-21R.

In one embodiment of the methods of the invention, the IL-21R-associateddisorder is selected from the group consisting of autoimmune disorders,inflammatory conditions, allergies, transplant rejections, andhyperproliferative disorders of the blood. In another embodiment, theIL-21R-associated disorder is selected from the group consisting ofimmune disorders, hyperproliferative disorders of the blood, transplantrejection, graft-versus-host disease, allergy (including atopicallergy), diabetes mellitus, arthritic disorders (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis, ankylosing spondylitis), spondyloarthropathy, multiplesclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosus (SLE), cutaneous lupus erythematosus, autoimmunethyroiditis, dermatitis (including atopic dermatitis, eczematousdermatitis), psoriasis, Sjögren's syndrome, IBD (including Crohn'sdisease, ulcerative colitis), asthma (including intrinsic asthma,allergic asthma), scleroderma and vasculitis. In a further embodiment,the IL-21R-associated disorder is selected from the group consisting ofmultiple sclerosis, systemic lupus erythematosus, psoriasis, transplantrejection, rheumatoid arthritis, and other arthritic disorders.

In one embodiment of the methods of the invention, the binding proteinor antigen-binding fragment thereof has an association constant forhuman IL-21R of at least 10⁵ M⁻¹s⁻¹. In another embodiment, the bindingprotein or antigen-binding fragment thereof inhibits IL-21-mediated BAF3cell proliferation with an IC₅₀ of about 1.75 nM or less, wherein theBAF3 cells comprise a human IL-21 receptor. In another embodiment, thebinding protein or antigen-binding fragment thereof inhibitsIL-21-mediated proliferation of TF1 cells with an IC₅₀ of about 14 nM orless, wherein the TF1 cells comprise a human IL-21 receptor. In stillanother embodiment, the binding protein or antigen-binding fragmentthereof inhibits IL-21-mediated proliferation of primary human B cellswith an IC₅₀ of about 1.9 nM or less, wherein the B cells comprise ahuman IL-21R. In yet another embodiment, the binding protein orantigen-binding fragment thereof inhibits IL-21-mediated proliferationof primary human CD4⁺ cells with an IC₅₀ of about 1.5 nM or less,wherein the CD4⁺ cells comprise a human IL-21R. In further embodimentsof the invention, other ranges and values for these parameters, and forother pharmacokinetic and pharmacodynamic parameters, are providedherein.

In one embodiment, the invention provides a method of determiningwhether an anti-IL-21R antibody is a therapeutic anti-IL-21R antibodycomprising the steps of: contacting a first blood sample from a subjectwith an IL-21 ligand; determining a level of expression of at least oneIL-21-responsive gene in the first blood sample contacted with the IL-21ligand; contacting a second blood sample from the subject with the IL-21ligand in the presence of an anti-IL-21R antibody; determining the levelof expression of the at least one IL-21-responsive gene in the secondblood sample contacted with the IL-21 ligand in the presence of theanti-IL-21R antibody; and comparing the levels of expression of the atleast one IL-21-responsive gene determined above, wherein a change inthe level of expression of the at least one IL-21-responsive geneindicates that the anti-IL-21R antibody is a therapeutic antibody. Inanother embodiment of the invention, the subject is a mammal, e.g., amonkey or a human. In another embodiment, the at least oneIL-21-responsive gene is selected from the group consisting of TNF,IFNγ, IL-6, IL-8, IL-10, CD19, STAT3, TBX21, CSF1, GZMB, PRF1, IL-2Rα,and IL-21R. In a further embodiment, the at least one IL-21-responsivegene is IL-2Rα.

In one embodiment, the invention provides a method of determining thepharmacodynamic activity of an anti-IL-21R antibody comprising detectinga modulation in a level of expression of at least one IL-21-responsivegene in a blood sample of a subject. In a further embodiment, detectingthe modulation in the level of expression of the at least oneIL-21-responsive gene comprises the steps of: administering theanti-IL-21R antibody to the subject, wherein the subject is treated withthe anti-IL-21R antibody; contacting a blood sample from the subjecttreated with the anti-IL-21R antibody with an IL-21 ligand; determiningthe level of expression of the at least one IL-21-responsive gene in theblood sample from the subject treated with the anti-IL-21R antibody andcontacted with the IL-21 ligand; and comparing the level of expressionof the at least one IL-21-responsive gene determined above with thelevel of expression of the at least one IL-21 responsive gene in adifferent blood sample contacted with the IL-21 ligand, wherein thedifferent blood sample is from a subject not treated with theanti-IL-21R antibody. In another embodiment of the invention, thesubject is a mammal, e.g., a monkey or a human. In another embodiment,the at least one IL-21-responsive gene is selected from the groupconsisting of TNF, IFNγ, IL-6, IL-8, IL-10, CD19, STAT3, TBX21, CSF1,GZMB, PRF1, IL-2Rα, and IL-21R. In a further embodiment, the at leastone IL-21-responsive gene is selected from the group consisting of CD19,GZMB, PRF1, IL-2Rα, IFNγ, and IL-6. In another further embodiment, theat least one IL-21-responsive gene is IL-2Rα.

In one embodiment, the invention provides a method of decreasing,inhibiting, or reducing an acute phase response in a subject, comprisingadministering to the subject a binding protein or antigen-bindingfragment thereof that specifically binds to human IL-21R in an amountsufficient to decrease, inhibit or reduce the acute phase response inthe subject, wherein the binding protein or antigen-binding fragmentthereof comprises at least one amino acid sequence that is at leastabout 95% identical to an amino acid sequence(s) selected from the groupconsisting of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,165-168, 171-193, 213-229, 240, 242, 244, 246, and 248. In a furtherembodiment, the binding protein or antigen-binding fragment thereof isadministered locally.

One embodiment of the invention is a method of increasing the efficacyof a vaccine formulation used to immunize a subject, comprisingadministering to the subject a therapeutically effective amount of abinding protein or antigen-binding fragment thereof that specificallybinds to human IL-21R, wherein the binding protein or antigen-bindingfragment thereof comprises at least one amino acid sequence that is atleast about 95% identical to an amino acid sequence(s) selected from thegroup consisting of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 165-168, 171-193, 213-229, 240, 242, 244, 246, and 248. Insome embodiments, the binding protein or antigen-binding fragmentthereof is administered before, during and/or after immunization.

Another embodiment of the invention provides a method for detecting thepresence of IL-21R in a sample in vitro, comprising (a) contacting asample with a binding protein or antigen-binding fragment thereof thatspecifically binds to human IL-21R, and (b) detecting formation of acomplex between the binding protein or antigen-binding fragment thereofand the sample, wherein a significant difference in the formation of thecomplex in the sample relative to in a control or reference sample orlevel is indicative of the presence of IL-21R in the sample, and whereinthe binding protein or antigen-binding fragment thereof comprises atleast one amino acid sequence that is at least about 95% identical to anamino acid sequence(s) selected from the group consisting of SEQ IDNOs:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165-168, 171-193,213-229, 240, 242, 244, 246, and 248. In a further embodiment, thesample is serum, plasma, or tissue.

Yet another embodiment of the invention provides a method for detectingthe presence of IL-21R in vivo, comprising (a) administering a bindingprotein or antigen-binding fragment thereof that specifically binds tohuman IL-21R to a subject under conditions that allow for binding of thebinding protein or antigen-binding fragment thereof to IL-21R, and (b)detecting formation of a complex between the binding protein orantigen-binding fragment thereof and IL-21R, wherein a significantdifference in the formation of the complex in the subject relative to acontrol or reference sample or level of formation of the complex isindicative of the presence of IL-21R, and wherein the binding protein orantigen-binding fragment thereof comprises at least one amino acidsequence that is at least about 95% identical to the amino acidsequence(s) selected from the group consisting of SEQ ID NOs:14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 165-168, 171-193, 213-229, 240, 242,244, 246, and 248. In some embodiments, the binding protein orantigen-binding fragment thereof is directly or indirectly labeled witha detectable substance to facilitate detection of bound or unboundantibody. In further embodiments, the detectable substance is an enzyme,a prosthetic group, a fluorescent material, a luminescent material, or aradioactive material.

In some embodiments, the invention provides methods further comprisingadministering to the subject another therapeutic agent chosen from thegroup consisting of a cytokine inhibitor, a growth factor inhibitor, animmunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, anenzyme inhibitor, a cytotoxic agent, and a cytostatic agent. In furtherembodiments, the therapeutic agent is chosen from the group consistingof a TNF antagonist, an IL-12 antagonist, an IL-15 antagonist, an IL-17antagonist, an IL-18 antagonist, an IL-19 antagonist, an IL-20antagonist, an IL-21 antagonist, an IL-23 antagonist, a T cell-depletingagent, a B cell-depleting agent, methotrexate, leflunomide, sirolimus(rapamycin) or an analog thereof, a cox2 inhibitor, a cPLA2 inhibitor,an NSAID, and a p38 inhibitor. As in other methods of the invention, insome embodiments the subject is a mammal, e.g., a human.

Another embodiment of the invention provides a method for measuring,determining, and/or assessing the levels of production of anti-productantibodies, e.g., anti-product antibodies to anti-IL-21R bindingproteins and antigen-binding fragments thereof.

Additional aspects of the disclosure will be set forth in part in thedescription, and in part will be obvious from the description, or may belearned by practicing the invention. The invention is set forth andparticularly pointed out in the claims, and the disclosure should not beconstrued as limiting the scope of the claims. The following detaileddescription includes exemplary representations of various embodiments ofthe invention, which are not restrictive of the invention as claimed.The accompanying figures constitute a part of this specification and,together with the description, serve only to illustrate embodiments andnot limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a-c) depicts the neutralization of proliferation of humanIL-21R-BaF3 cells by scFv. Cells were mixed with the indicated scFv andthen incubated with 100 pg/ml of human IL-21. Proliferation was measuredby CELLTITER-GLO® (Promega Corporation, Madison, Wis.) after 48 hr.

FIG. 2( a-c) depicts the neutralization of proliferation of humanIL-21R-TF1 cells by scFv. Cells were mixed with the indicated scFv andthen incubated with 100 pg/ml of human IL-21. Proliferation was measuredby CELLTITER-GLO® after 48 hr.

FIG. 3( a-c) depicts the neutralization of proliferation of murineIL-21R-BaF3 cells by scFv. Cells were mixed with the indicated scFv andthen incubated with 400 pg/ml of murine IL-21. Proliferation wasmeasured by CELLTITER-GLO® after 48 hr.

FIG. 4( a-c) depicts scFv competition with parental antibody 18A5 IgGfor binding to murine IL-21R. The scFv were mixed withbiotinylated-murine IL-21R-H/F, and the mixtures were added to antibody18A5 immobilized on an ELISA plate. Capture of mIL-21R was detected withHRP-streptavidin, and competition for binding to mIL-21R was indicatedby a reduction in the A450 signal.

FIG. 5( a-i) depicts the neutralization of IL-21-dependent proliferationby 21 heavy chain/light chain pairs. Antibodies, as indicated in thefigure, were added to cells. IL-21 was subsequently added, andproliferation measured with CELLTITER-GLO® after 48 hr. Assays wereconducted on human IL-21R-BaF3 cells with 100 pg/ml of human IL-21(FIGS. 5 a-c), human IL-21R-TF1 cells with 100 pg/ml of human IL-21(FIGS. 5 d-f), or murine IL-21R-BaF3 cells with 400 pg/ml of murineIL-21 (FIGS. 5 g-i).

FIG. 6( a-l) depicts the binding of 21 anti-IL-21R IgGs to CHO cellstransiently expressing human IL-21R (FIGS. 6 a-c), rat IL-21R (FIGS. 6d-f), cynomolgus monkey IL-21R (FIGS. 6 g-i), and human gamma commonchain (FIGS. 6 j-1). CHO cells were transiently transfected with IL-21Ror the control gamma common chain, and binding was detected withHRP-conjugated anti-human IgG in a cell-based ELISA.

FIG. 7( a-c) depicts the binding specificity of particular anti-IL-21Rantibodies (FIG. 7 a, AbS; FIG. 7 b, AbQ, AbT, AbO; FIG. 7 c, AbR, AbP,and AbU), measured by surface plasmon resonance. The anti-IL-21Rantibodies were captured on anti-human IgG, and subsequent binding toeither murine IL-21R-H/F, human IL-13-H/F, human IL-2Rβ, or humansoluble IL-4R was measured in a BIACORE™ instrument (GE Healthcare,Piscataway, N.J.). FIG. 7 d shows that human IL-2Rβ and human solubleIL-4R are captured by specific anti-IL-2Rβ and anti-IL-4R antibodies,respectively (control).

FIG. 8( a-d) depicts the binding of anti-IL-21R antibodies to human andmurine IL-21R. The indicated human anti-IL-21R antibodies were capturedon anti-human IgG immobilized on a BIACORE™ chip. Varying concentrationsof human IL-21R-His/FLAG (FIGS. 8 a-b) and murine IL-21R-His/FLAG (FIGS.8 c-d) were allowed to flow over the chip, and binding and dissociationwere monitored.

FIG. 9( a-d) depicts the binding of anti-IL-21R antibodies to human andcynomolgus monkey IL-21R. Human anti-IL-21R antibodies AbS and AbT werecaptured on anti-human IgG immobilized on a BIACORE™ chip. Varyingconcentrations of human and cynomolgus monkey IL-21R-His/FLAG wereallowed to flow over the chip, and binding and dissociation weremonitored. FIG. 9 a shows cynomolgus monkey IL-21R-His/FLAG binding toAbS. FIG. 9 b shows human IL-21R-His/FLAG binding to AbS. FIG. 9 c showscynomolgus monkey IL-21R-His/FLAG binding to AbT. FIG. 9 d shows humanIL-21R-His/FLAG binding to AbT.

FIG. 10( a-b) depicts an epitope assessment of IL-21R antibodies. In theexperiment depicted in FIG. 10 a (see also illustration at left ofY-axis), murine IL-21R-H/F (His-Flag fusion protein) was captured byanti-IL-21R antibody AbS immobilized on a BIACORE™ chip. Additionalanti-IL-21R antibodies (AbS, AbT, D5 (D5-20, a neutralizing anti-murineIL-21R antibody), and 7C2 (a normeutralizing anti-murine IL-21R controlantibody)) were flowed over the chip and their binding to the capturedIL-21R-H/F was monitored. In the experiment depicted in FIG. 10 b, humanIL-21R-H/F was captured by anti-IL-21R antibody AbS immobilized on aBIACORE™ chip. Additional anti-IL-21R antibodies (AbS, AbT, and 9D2 (anormeutralizing anti-human IL-21R control antibody)) were flowed overthe chip and their binding to the captured IL-21R-H/F was monitored.

FIG. 11( a-c) depicts the neutralization of proliferation of humanIL-21R-BaF3 cells and murine IL-21R-BaF3 cells by the indicatedantibodies. Antibodies were added to cells. IL-21 was subsequently addedand proliferation measured with CELLTITER-GLO® after 48 hr. Assays wereconducted on human IL-21R-BaF3 cells with 100 pg/ml of human IL-21 (FIG.11 a), murine IL-21R-BaF3 cells with 200 pg/ml of murine IL-21 (FIG. 11b), and human IL-21R-TF1 cells with 100 pg/ml of human IL-21 (FIG. 11c).

FIG. 12( a-b) depicts the neutralization of IL-21-dependentproliferation of human primary B cells. The indicated antibodies wereadded to primary human B cells along with anti-CD40 antibodies and humanIL-21. Incorporation of ³H-thymidine was measured after three days. FIG.12 a depicts the comparison between AbQ, AbR, AbS, AbT, AbU, IL-13triple-mutant, and 18A5 parental antibody; FIG. 12 b depicts thecomparison between AbT, AbV, AbW, AbU, and human IgG1 control (hIg1).

FIG. 13 depicts the neutralization of IL-21-dependent proliferation ofhuman primary CD4⁺ T cells. The indicated antibodies were added toactivated primary human CD4⁺ T cells along with human IL-21, andincorporation of ³H-thymidine was measured after three days.

FIG. 14 depicts the neutralization of IL-21-dependent proliferation ofmurine primary CD8⁺ T cells. The indicated antibodies were added toactivated primary murine CD8⁺ T cells along with human IL-21, andincorporation of ³H-thymidine was measured after three days.

FIG. 15 depicts the measurement of antibody-dependent cellularcytotoxicity (ADCC) induced by anti-IL-21R antibodies. PBMC-dependentkilling of CFSE-labeled BJAB cells coated with the indicated anti-IL-21Rantibodies was measured by incorporation of propidium iodide. Theanti-CD20 antibody rituximab (RITUXAN®, Genentech, Inc., South SanFrancisco, Calif.) was included as a positive control, and an anti-IL-13antibody was included as a negative control.

FIG. 16 depicts complement C1q binding by anti-IL-21R antibodies. Theindicated anti-IL-21R antibodies were immobilized on an ELISA plate and,following incubation with human serum, C1q binding was measured withchicken anti-human C1q and an HRP-conjugated anti-chicken IgY antibody.The anti-CD20 antibody rituximab (RITUXAN®) was included as a positivecontrol, and an anti-IL-13 antibody was included as a negative control.

FIG. 17( a-c) depicts amino acid sequences for AbQ, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 18( a-c) depicts amino acid sequences for AbR, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 19( a-c) depicts amino acid sequences for AbW, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 20( a-c) depicts amino acid sequences for AbS, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 21( a-c) depicts amino acid sequences for AbT, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 22( a-c) depicts amino acid sequences for AbO, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 23( a-c) depicts amino acid sequences for AbP including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 24( a-c) depicts amino acid sequences for AbU, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 25( a-c) depicts amino acid sequences for AbV, including V_(H) andV_(L) domains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIGS. 26( a-g) depict results generated from additional studies thatwere performed similarly to those performed to generate the resultsshown in FIGS. 5, 11, 12, 13, and 14 (described above).

FIG. 27 a depicts IL-21 cytokine competition with antibody AbT forbinding to murine IL-21R. Vehicle, or increasing amounts of IL-21, wasmixed with biotinylated murine IL-21R-His/FLAG, and the mixtures wereadded to AbT immobilized on an ELISA plate. Capture of mIL-21R wasdetected with HRP-streptavidin, and competition for binding to mIL-21Rwas indicated by a reduction in the A450 signal. FIG. 27 b depicts thecompetition of AbS and IL-21 for binding to murine IL-21R. AbS andmIL-21 were mixed and applied to immobilized mIL-21R-Fc in ELISA plates,and the binding of AbS (filled diamonds), isotype control antibody (opentriangles), or mIL-21 (squares) was monitored.

FIG. 28 depicts IL-21-dependent ³H thymidine incorporation into rat CD3T cells in the presence of anti-IL-21R antibodies AbS, AbU, AbV, or AbW,or an isotype control antibody, hIgG1-TM.

FIG. 29( a-b) depicts binding of AbS to either of two isoforms of rabbitIL-21R-Fc presented by immobilized anti-mouse IgG antibodies in theabsence (FIG. 29 a) or presence (FIG. 29 b) of 10% conditioned mediumcontaining rabbit IL-21.

FIG. 30( a-h) depicts NP-specific primary antibody response in animalstreated with anti-IL-21R antibodies. FIG. 30 a depicts NP-specific IgGresponse and FIG. 30 b depicts NP-specific IgM response. FIGS. 30 c-fdepict the NP-specific IgG subclass responses in repeats of theexperiment shown in FIG. 30 a, with ELISA data shown for individualanimals: total IgG (FIG. 30 c), IgG1 (FIG. 30 d), IgG2a (FIG. 30 e), andIgG2c (FIG. 30 f). FIG. 30 g depicts NP-specific IgG response over atwo-month period. FIG. 30 h depicts NP-specific IgG antibody-secretingcells (ASC) found in bone marrow at the end of the two-month studyperiod.

FIG. 31( a-g) depicts anti-double-stranded DNA (anti-dsDNA) antibodiesin MRL-Fas^(lpr) mice treated with anti-IL-21R antibodies.Twelve-week-old male MRL-Fas^(lpr) mice were treated with either saline(control) or the indicated triple-mutant antibodies (10 mg/kg i.p.,3×/week); an anti-human IL-13 human IgG1 A234 A235 A237 triple-mutantantibody with no reactivity to murine IL-21 (IL-13™) was used as anisotype control. Serum collected biweekly was tested by ELISA for thepresence of anti-dsDNA. FIG. 31 a shows anti-dsDNA antibody titersfollowing treatment. FIG. 31 b shows prebleed anti-dsDNA antibodytiters. FIG. 31 c shows anti-dsDNA antibody titers after two weeks ofdosing. FIG. 31 d shows anti-dsDNA antibody titers after four weeks ofdosing. FIG. 31 e shows anti-dsDNA antibody titers after six weeks ofdosing. FIG. 31 f shows anti-dsDNA antibody titers after eight weeks ofdosing. FIG. 31 g shows anti-dsDNA antibody titers after ten weeks ofdosing. In FIGS. 31 a and 31 c-g, asterisks indicate a significantdifference as compared to both the saline and IL-13 controls (p<0.01).In FIG. 31 b, the asterisk indicates a significant difference ascompared to the other three groups (p<0.01). The dashed line in FIGS. 31b-g indicates the level of detection.

FIG. 32( a-b) depicts IgG deposits in kidneys of MRL-Fas^(lpr) micetreated with anti-IL-21R antibodies. Twelve-week-old male MRL-Fas^(lpr)mice were treated (10 mg/kg i.p., 3×/week) with either saline (control)or the indicated triple-mutant antibodies; anti-human IL-13 human IgG1A234 A235 A237 triple-mutant antibody with no reactivity to murine IL-21was used as an isotype control. Following 10 weeks of treatment, micewere sacrificed and IgG deposits in the kidneys were identified byimmunocytochemistry (FIG. 32 b; glomeruli are indicated by dashedcircles; examples of IgG deposits are indicated by arrowheads). Stainingintensity was scored on a scale of 1-5 (FIG. 32 a).

FIG. 33( a-b) depicts IgM and complement C3 deposits in kidneys ofMRL-Fas^(lpr) mice treated with anti-IL-21R antibodies. Twelve-week-oldmale MRL-Fas^(lpr) mice were treated (10 mg/kg i.p., 3×/week) witheither saline (control) or the indicated triple-mutant antibodies.Following ten weeks of treatment, mice were sacrificed and IgM (FIG. 33a) and complement C3 (FIG. 33 b) deposits in the kidneys were identifiedby immunocytochemistry. Staining intensity was scored on a scale of 1-5.

FIG. 34( a-c) depicts IgG, IgM and complement C3 deposits in brains ofMRL-Fas^(lpr) mice treated (10 mg/kg i.p., 3×/week) with anti-IL-21Rantibodies. Twelve-week-old male MRL-Fas^(lpr) mice were treated witheither saline (control) or the indicated triple-mutant antibodies.Following ten weeks of treatment, mice were sacrificed and IgG (FIG. 34a), IgM (FIG. 34 b), and complement C3 (FIG. 34 c) deposits in the brainwere identified by immunocytochemistry. Staining intensity was scored ona scale of 1-5.

FIG. 35( a-c) depicts lymphocytic infiltrates in kidneys ofMRL-Fas^(lpr) mice treated with anti-IL-21R antibodies. Twelve-week-oldmale MRL-Fas^(lpr) mice were treated (10 mg/kg i.p., 3×/week) witheither saline (control) or the indicated triple-mutant antibodies.Following ten weeks of treatment, mice were sacrificed andhematoxylin/eosin (H/E)-stained kidney sections were examined forlymphocyte infiltration in three zones, cortex interstitium (a supportstructure for the glomeruli) (FIG. 35 a), cortex perivascular (FIG. 35b), and peripelvic (near the origin of the ureter) (FIG. 35 c).Lymphocyte numbers were scored on a scale of 1-5.

FIG. 36 depicts lymphocytic infiltrates in lungs of MRL-Fas^(lpr) micetreated with anti-IL-21R antibodies. Twelve-week-old male MRL-Fas^(lpr)mice were treated (10 mg/kg i.p., 3×/week) with either saline (control)or the indicated triple-mutant antibodies. Following ten weeks oftreatment, mice were sacrificed and H/E-stained lung sections wereexamined for lymphocyte infiltration. Lymphocyte numbers were scored ona scale of 1-5.

FIGS. 37 a-b depict the mouse anti-human antibody (MAHA) response inMRL-Fas^(lpr) mice treated with anti-IL-21R antibodies. Twelve-week-oldmale MRL-Fas^(lpr) mice were treated (10 mg/kg i.p., 3×/week) with theindicated triple-mutant antibodies. Serum, collected biweekly, wastested by ELISA for the presence of murine antibodies capable of bindingto the same human antibodies with which the mice were treated (FIG. 37a), or for the presence of murine antibodies capable of binding to theother anti-IL-21 human antibodies (FIG. 37 b). Asterisks in FIG. 37 aindicate a significant difference as compared to the anti-IL-13-treatedgroup (p<0.05).

FIG. 38( a-d) depicts development of anti-dsDNA antibodies inMRL-Fas^(lpr) mice treated with anti-IL-21R antibodies. FIG. 38 adepicts anti-dsDNA antibody titer for mice treated with AbS, and FIG. 38b depicts anti-dsDNA antibody titer for mice treated with AbT. FIGS. 38c-d represent kidney pathology and renal inflammatory foci inMRL-Fas^(lpr) mice treated with AbS and AbT.

FIG. 39( a-j) depicts cellular infiltration into the dorsal air pouch inmice or rats treated with anti-IL-21R antibodies. FIGS. 39 a-e: threedays after injection of 3 ml of air under the dorsal skin of BALB/C miceto create an air pouch, pouches were reinflated. Two days later, eithersaline (control) or the indicated triple-mutant antibodies were injectedi.p. One hundred ng of murine IL-21 was injected into the air pouch 24hr after antibody injection. Six hours later, the pouches were washedout with 3 ml of PBS, and total cell counts (FIG. 39 a), monocytes (FIG.39 b), lymphocytes (FIG. 39 c), and neutrophils (FIG. 39 d) weredetermined. FIG. 39 e depicts total cell counts in a replicate of theexperiment shown in FIGS. 39 a-d. FIGS. 39 f-j depict total cellinfiltration into air pouches in rats following treatment with mouseIL-21 and anti-IL-21R antibodies. FIG. 39 f depicts cellularinfiltration following administration of either 20 μg mIL-21 for 6 hr or1 μg mIL-21 for 20 hr, in the presence or absence of AbS or controlantibody. FIGS. 39 g-h depict cellular infiltration into rat air pouchesin replicate experiments in which 20 μg mIL-21 was administered for 6 hrin the presence of 1, 3, or 10 μg/kg AbS or a control antibody. In theexperiment shown in FIG. 39 i, rats were treated for 6 hr with 20 μgmIL-21 and 1 mg/kg antibody (either AbS or isotype control), singly orin combination, as well as a combination of 20 μg mIL-21 and 10 mg/kgantibody. In FIG. 39 j, 10, 20, or 40 μg mIL-21 were tested incombination with either 1 or 10 mg/kg AbS.

FIG. 40 depicts the concentration-time profiles of AbS in CD-1 miceafter a single intravenous or subcutaneous administration.Concentrations below the limit of quantitation were treated as zero forthe calculation of mean and standard deviation. N=4-8 for each datapoint.

FIG. 41 depicts the concentration-time profiles of AbS in DBA andMRL-Fas^(lpr) mice after a single intraperitoneal administration.Concentrations below the limit of quantitation were treated as zero forthe calculation of mean and standard deviation. N=4-8 for each datapoint.

FIGS. 42 a and b depict the observed and predicted AbS concentrations inMRL-Fas^(lpr) mice after multiple i.p. administrations at 10, 5, and 2.5mg/kg doses, 3×/week, for ten weeks. Concentrations below the limit ofquantitation were treated as zero for the calculation of mean and SD.N=7-8 per time point.

FIGS. 43 a and b depict the concentration-time profiles of AbS incynomolgus monkeys after administrations as shown. Concentrations belowthe limit of quantitation (<30 ng/ml) were treated as zero for thecalculation of mean and standard deviation. N=3 for each group. SAN inFIG. 43 a is the study animal number.

FIG. 44( a-c) depicts the concentration-time profiles of AbS and AbTafter a single administration to mice. FIG. 44 a shows a profile after a10 mg/kg i.v. administration in CD-1 mice. FIG. 44 b shows a profileafter a 10 mg/kg i.p. administration in MRL-Fas^(lpr) mice. FIG. 44 cshows a profile after an 8 mg/kg i.p. administration in DBA mice.Concentrations below the limit of quantitation were treated as zero forthe calculation of mean and standard deviation. N=4-8 for each datapoint.

FIG. 45 depicts the observed and predicted AbT concentrations inMRL-Fas^(lpr) mice after multiple i.p. administrations at 20 mg/kgdoses, 3×/week, for ten weeks. Concentrations below the limit ofquantitation were treated as zero for the calculation of mean andstandard deviation. N=6-8 per time point.

FIGS. 46 a and b depict the concentration-time profiles of AbT and AbSin cynomolgus monkeys after administrations as shown. Concentrationsbelow the limit of quantitation (<30 ng/ml) were treated as zero for thecalculation of mean and standard deviation. N=3 for each group.

FIG. 47 depicts the concentration-time profiles of the anti-IL-21Rantibodies AbS, AbT, and ¹²⁵I-D5 (8 mg/kg, i.p.) in male DBA mice. Humanantibodies AbS and AbT were quantitated by anti-human IgG ELISA, and the¹²⁵I-labeled murine antibody D5 was quantitated by monitoring theradiolabel.

FIG. 48 a depicts mean serum concentrations (Y-axis; ng/ml) of AbS, AbT,and an isotype control in Sprague-Dawley (S-D) rats after a single 10mg/kg i.v. dose. FIG. 48 b depicts anti-IL-21R antibodies AbS-AbW aftera single 10 mg/kg i.v. dose in S-D rats. Individual data points <LOQ (45ng/mL) were treated as zero for calculations of mean and SD. If meanvalue was below LOQ (e.g., when all animals had value <LOQ), data pointsare not shown.

FIG. 49 depicts means serum concentrations (Y-axis; ng/ml) of AbS in S-Drats after a single i.v., i.p., or s.c. dose.

FIG. 50( a-d) depicts the biodistribution of ¹²⁵I-AbS in IL-21R knockoutand wild-type C57BL/6 (control) mice after a single 2.5 mg/kgintravenous dose.

FIG. 50 a shows the concentration of ¹²⁵I-AbS in tissues in controlC57BL/6 mice.

FIG. 50 b shows the concentration of ¹²⁵I-AbS in IL-21R knockout mice.FIG. 50 c shows the serum concentration of ¹²⁵I-AbS in control C57BL/6and IL-21R knockout mice. FIG. 50 d shows cumulative urine counts of¹²⁵I-AbS in control C57BL/6 and IL-21R knockout mice. Concentrationsbelow LOQ were treated as zero for the calculation of mean and SD.N=9-10 for each serum or tissue data point; N=5-10 for each urine datapoint.

FIG. 51( a-b) depicts the biodistribution of ¹²⁵I-AbS in MRL-Fas^(lpr)mice after a single 2.5 mg/kg i.p. dose. FIG. 51 a shows theconcentration of ¹²⁵I-AbS in tissues in MRL-Fas^(lpr) mice. FIG. 51 bshows urine counts of ¹²⁵I-AbS in MRL-Fas^(lpr) mice.

FIG. 52 depicts the serum concentration of ¹²⁵I-AbS in MRL-Fas^(lpr)mice after single 2.5 and 10 mg/kg i.p doses. Concentrations below LOQwere treated as zero for the calculation of mean and SD. N=4-8 for eachdata point.

FIG. 53 a depicts percent inhibition (Y-axis) of IL-21-induced IL-2Rα(IL2RA), PRF1, IL-6, and CD19 expression upon increasing concentrationsof AbS (X-axis) in human whole blood samples, calculated fromfold-change; FIG. 53 b depicts inhibition of IL-21-induced relativeexpression level (RQ; Y-axis) of IL-6 in response to increasing AbSconcentrations (X-axis; concentration Ab (nM)) in human whole bloodsamples.

FIG. 54 depicts the genes included on custom TLDA (Taqman® Low DensityArray) for assay; Endogenous (Endo) controls are indicated.

FIG. 55 demonstrates relative quantification (RQ; Y-axis) of geneexpression of six examined genes (CD19, GZMB, IFNγ (IFNG), IL-2Rα(IL2RA), IL-6, and PRF1) at different concentrations of IL-21 at either2, 4, 6, or 24 hr time points (X-axis).

FIG. 56 a demonstrates relative quantification (RQ; Y-axis) of geneexpression of six examined genes (CD19, GZMB, IFNγ, IL-2Rα, IL-6, andPRF1) after incubation with IL-21 and different concentrations of eitherAbS or IgG1TM control (X-axis). FIGS. 56 b-c depict percent inhibition(Y-axes) of IL-21 response of the same genes after treatment withdifferent concentrations of either AbS or control IgG1TM (X-axes).

FIG. 57( a-c) depicts distribution of Relative Quantification (RQ)values for IL-2Rα gene expression in whole blood of male cynomolgusmonkeys following ex vivo stimulation with recombinant human IL-21. FIG.57 a demonstrates the histogram analysis (Y-axis) for the RQ values(X-axis), and FIG. 57 b demonstrates histogram analysis (Y-axis) for thelog₂-transformed RQ values (X-axis), which were obtained using astatistical software package. The solid line represents Gaussiandistribution. The dotted line represents cutoff RQ for the enrollmentinto the in vivo study, defined using the formula: log{RQ_(cutoff)}=mean of the log-transformed RQ values−SD of thelog-transformed RQ values. FIG. 57 c compares individual animal andmedian (solid lines) RQ values (Y-axis) of whole blood aliquotsstimulated with IL-21 and AbT (triangles) or IL-21 and control IgG(squares) as compared to those with IL-21 stimulation alone (circles).

FIG. 58 depicts serum concentrations (Y-axis) following single 10 mg/kgi.v. administration of anti-IL-21R antibody AbS (“A”) or AbT (“B”) tomale cynomolgus monkeys, collected up to day 148 for AbS and up to day36 for AbT (X-axis). Data points with serum concentrations below thelower LOQ (30 ng/mL) are not shown.

FIG. 59( a-c) shows correlation of antibody serum concentrations (secondY-axis), pharmacodynamic (PD) activity (“RQ”; first Y-axis), andanti-product antibody response (indicated by “A”), includingneutralizing anti-product antibody response (indicated by “AN”),following single 10 mg/kg i.v. administration of anti-IL-21R antibodyAbS to male cynomolgus monkeys, as measured at various times pre- andpost-dose administration (X-axis; Time(days)). FIGS. 59 a-c depictresults for Animals 1-3, respectively.

FIG. 60( a-c) shows correlations similar to those in FIG. 59 for AbT(single 10 mg/kg i.v. administration). FIGS. 60 a-c depict results forAnimals 4-6, respectively.

FIG. 61 depicts the concentration-time profiles of AbS-AbW in cynomolgusmonkeys after a single i.v. administration. Concentrations below thelimit of quantitation (<30 ng/ml) were treated as zero for calculations.For AbS (filled circles, Study 1; open diamonds, Study 2), AbT (opencircles), AbV (filled diamonds), and AbU (open triangles), n=3; 10 mg/kgdose. For AbW (open squares), n=2; 1 mg/kg dose.

FIG. 62( a-b) depicts individual concentrations (ng/ml) of AbS afterthree weekly i.v. administrations (arrows) of 2 (FIG. 62 a) or 10 mg/kg(FIG. 62 b) to tetanus-toxoid challenged male and female cynomolgusmonkeys.

FIG. 63( a-b) depicts allometric scaling of AbS PK parameters after i.v.administration. Body weight (W); maximum span potential (in years; MLP).FIG. 63 a represents CL·MLP (Y-axis) plotted against body weight(X-axis);

FIG. 63 b represents volume of distribution (Y-axis) plotted againstbody weight (X-axis). Solid lines represent fitted curves based on alinear regression using data from mice, rats, and cynomolgus monkeys.Dotted lines represent 95% confidence intervals.

FIG. 64( a-b) represents allometric scaling of AbT PK parameters afteri.v. administration. Body weight (W); brain weight (“BW”). FIG. 64 arepresents CL*BW (Y-axis) plotted against body weight (X-axis); FIG. 64b represents volume of distribution (Y-axis) plotted against body weight(X-axis).

FIG. 65( a-b) depicts the effects of AbS on the development ofproteinuria (FIG. 65 a) and anti-dsDNA IgG serum antibody titers (FIG.65 b) in NZBWF1/J mice. Female 26-week old NZBWF1/J mice wereadministered 400 μg of either saline, anti-E.tenalla antibody,mCTLA-4Ig, hIgGTm antibody or AbS antibody 3×/week for 10 weeks. Urineprotein levels and anti-dsDNA IgG serum antibody titers were measured atthe beginning of the study, and every two weeks thereafter for 10 weeks.Animals treated with CTLA4-Ig showed significant reductions inanti-dsDNA titers (asterisks; p<0.05) at weeks 4-10.

FIG. 66( a-c) depicts the effects of anti-IL-21R neutralization ondisease outcome in a semi-therapeutic CIA mouse model of rheumatoidarthritis. Female DBA/1 mice were immunized and boosted with bovinecollagen type II; when 10% of the animals in the study exhibited pawswelling, mice were dosed 3×/week for 30 days with 8 mg/kg of eithermurine IgG2a isotype control antibody, anti-mouse IL-21R antibody D5(murine IgG2a antibody), mTNFRII-Fc (positive control, murine IgG2aisotype) (FIG. 66 a); or anti-IL-13TM antibody (human IgG1 isotypecontrol antibody), AbT, or AbS (FIG. 66 b). Individual animal scores onday 30 of the study are depicted in FIG. 66 c.

FIG. 67( a-b) depicts the development of amnestic tetanus-specific IgMand IgG serum antibody responses to tetanus toxoid in cynomolgus monkeysin the presence of AbS. Nine male and nine female cynomolgus monkeyswere immunized with tetanus toxoid and rested for 43 days. After 43days, male and female monkeys were randomly assigned into groups ofthree, and treated with either saline (vehicle), 2 mg/kg AbS, or 10mg/kg AbS 1×/week for 3 weeks. Twenty-four hr after the first dose ofeither vehicle or AbS (arrows), monkeys were immunized a second timewith tetanus toxoid. Monkeys were routinely bled throughout the courseof the study, and examined for tetanus-specific IgM (FIG. 67 a) and IgG(FIG. 67 b) serum antibody titers.

DETAILED DESCRIPTION OF THE INVENTION

The binding proteins of the present invention were initially derivedfrom parental antibody 18A5, but differ from 18A5 in the amino acidsequences of portions of the heavy chain and/or light chaincomplementarity determining region 3 (CDR3). Additionally, the presentbinding proteins show improved potency in binding to and neutralizingboth human and murine IL-21R as compared to 18A5 in the equivalentformat (e.g., scFv or IgG). High-potency neutralization of IL-21R fromboth species (human and mouse) by a single binding protein has notpreviously been reported. The present binding proteins having a greaterneutralization potency than their parental antibody may translate intohigher efficacy as compared to agents previously described. In addition,the amino acid sequence of the V_(H) and V_(L) framework regions hasbeen altered to match sequences encoded by human genomic sequence,thereby reducing the potential for human anti-human antibody responsesin patients treated with the present binding proteins.

DEFINITIONS

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description and elsewhere in the specification.

The term “binding protein” as used herein includes any naturallyoccurring, recombinant, synthetic, or genetically engineered protein, ora combination thereof, that binds an antigen, target protein, orpeptide, or a fragment(s) thereof. Binding proteins of the invention caninclude antibodies, or be derived from at least one antibody fragment.The binding proteins can include naturally occurring proteins and/orproteins that are synthetically engineered. Binding proteins of theinvention can bind to an antigen or a fragment thereof to form a complexand elicit a biological response (e.g., agonize or antagonize aparticular biological activity). Binding proteins can include isolatedantibody fragments, “Fv” fragments consisting of the variable regions ofthe heavy and light chains of an antibody, recombinant single-chainpolypeptide molecules in which light and heavy chain variable regionsare connected by a peptide linker (“scFv proteins”), and minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region. Binding protein fragments can also includefunctional fragments of an antibody, such as, for example, Fab, Fab′,F(ab′)₂, Fc, Fd, Fd′, Fv, and a single variable domain of an antibody(dAb). The binding proteins can be double or single chain, and cancomprise a single binding domain or multiple binding domains.

Binding proteins can also include binding domain-immunoglobulin fusionproteins, including a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge or hinge-acting regionpolypeptide, which in turn is fused or otherwise connected to a regioncomprising one or more native or engineered constant regions from animmunoglobulin heavy chain other than CH1, for example, the CH2 and CH3regions of IgG and IgA, or the CH3 and CH4 regions of IgE (see, e.g.,Ledbetter et al., U.S. Patent Publication 2005/0136049, for a morecomplete description). The binding domain-immunoglobulin fusion proteincan further include a region that includes a native or engineeredimmunoglobulin heavy chain CH2 constant region polypeptide (or CH3 inthe case of a construct derived in whole or in part from IgE) that isfused or otherwise connected to the hinge region polypeptide, and anative or engineered immunoglobulin heavy chain CH3 constant regionpolypeptide (or CH4 in the case of a construct derived in whole or inpart from IgE) that is fused or otherwise connected to the CH2 constantregion polypeptide (or CH3 in the case of a construct derived in wholeor in part from IgE). Typically, such binding domain-immunoglobulinfusion proteins are capable of at least one immunological activityselected from the group consisting of antibody-dependent cell-mediatedcytotoxicity, complement fixation, and/or binding to a target, forexample, a target antigen. The binding proteins of the invention can bederived from any species including, but not limited to mouse, rat,human, camel, llama, fish, shark, goat, rabbit, chicken, and bovine.

The term “antibody” as used herein refers to an immunoglobulin that isreactive to a designated protein or peptide or fragment thereof.Suitable antibodies include, but are not limited to, human antibodies,primatized antibodies, chimeric antibodies, monoclonal antibodies,monospecific antibodies, polyclonal antibodies, polyspecific antibodies,nonspecific antibodies, bispecific antibodies, multispecific antibodies,humanized antibodies, synthetic antibodies, recombinant antibodies,hybrid antibodies, mutated antibodies, grafted conjugated antibodies(i.e., antibodies conjugated or fused to other proteins, radiolabels,cytotoxins), and in vitro-generated antibodies. The antibody can be fromany class of antibodies including, but not limited to, IgG, IgA, IgM,IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4)of antibodies. The antibody can have a heavy chain constant regionchosen from, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody can also havea light chain chosen from, e.g., kappa (κ) or lambda (λ). The antibodiesof the invention can be derived from any species including, but notlimited to mouse, rat, human, camel, llama, fish, shark, goat, rabbit,chicken, and bovine. Constant regions of the antibodies can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function). Typically, the antibody specifically binds to apredetermined antigen, e.g., an antigen associated with a disorder,e.g., an inflammatory, immune, autoimmune, neurodegenerative, metabolic,and/or malignant disorder.

The term “single domain binding protein” as used herein includes anysingle domain binding scaffold that binds to an antigen, protein, orpolypeptide. Single domain binding proteins can include any natural,recombinant, synthetic, or genetically engineered protein scaffold, or acombination thereof, that binds an antigen or fragment thereof to form acomplex and elicit a biological response (e.g., agonize or antagonize aparticular biological activity). Single domain binding proteins may bederived from naturally occurring proteins or antibodies, or they can besynthetically engineered or produced by recombinant technology. Singledomain binding proteins may be any in the art or any future singledomain binding proteins, and may be derived from any species including,but not limited to mouse, human, camel, llama, fish, shark, goat,rabbit, chicken, and bovine. In some embodiments of the invention, asingle domain binding protein scaffold can be derived from a variableregion of the immunoglobulin found in fish, such as, for example, thatwhich is derived from the immunoglobulin isotype known as Novel AntigenReceptor (NAR) found in the serum of shark. Methods of producing singledomain binding scaffolds derived from a variable region of NAR(“IgNARs”) are described in International Application Publication No. WO03/014161 and Streltsov (2005) Protein Sci. 14(11):2901-09.

In other embodiments, a single domain binding protein is a naturallyoccurring single domain binding protein, which has been described in theart as a heavy chain antibody devoid of light chains. Such single domainbinding proteins are disclosed in, e.g., International ApplicationPublication No. WO 94/004678. For clarity reasons, a variable domainbinding protein that is derived from a heavy chain antibody naturallydevoid of light chain is known herein as a VHH or “nanobody” todistinguish it from the conventional VH of four-chain immunoglobulins.Such a VHH molecule can be derived from antibodies raised in Camelidaespecies, for example in camel, llama, dromedary, alpaca, and guanaco.Other families besides Camelidae may also be used to produce heavy chainbinding proteins naturally devoid of light chains. VHH molecules areapproximately ten times smaller than traditional IgG molecules. They aresingle polypeptides and are very stable, resisting extreme pH andtemperature conditions. Moreover, they are resistant to the action ofproteases, which is not the case for conventional antibodies.Furthermore, in vitro expression of VHHs can produce high-yield,properly folded functional VHHs. In addition, binding proteins generatedin Camelids can recognize epitopes other than those recognized byantibodies generated in vitro via antibody libraries or via immunizationof mammals other than Camelids (see, e.g., International ApplicationPublication Nos. WO 97/049805 and WO 94/004678, both hereby incorporatedby reference herein).

The terms “antigen-binding domain” and “antigen-binding fragment” referto a part of a binding protein that comprises amino acids responsiblefor the specific binding between the binding protein and an antigen. Thepart of the antigen that is specifically recognized and bound by thebinding protein is referred to as the “epitope.” An antigen-bindingdomain may comprise a light chain variable region (V_(L)) and a heavychain variable region (V_(H)) of an antibody; however, it does not haveto comprise both. Fd fragments, for example, have two V_(H) regions andoften retain antigen-binding function of the intact antigen-bindingdomain. Examples of antigen-binding fragments of a binding proteininclude, but are not limited to: (1) a Fab fragment, a monovalentfragment having V_(L), V_(H), C_(L) and C_(H)1 domains; (2) a F(ab′)₂fragment, a bivalent fragment having two Fab fragments linked by adisulfide bridge at the hinge region; (3) an Fd fragment, having twoV_(H) and one C_(H)1 domains; (4) an Fv fragment, having the V_(L) andV_(H) domains of a single arm of an antibody; (5) a dAb fragment (see,e.g., Ward et al. (1989) Nature 341:544-46), having a V_(H) domain; (6)an isolated CDR; and (7) a single chain variable fragment (scFv).Although the two domains of an Fv fragment, V_(L) and V_(H) are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as scFv) (see, e.g., Bird et al. (1988) Science242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83).These binding domain fragments can be obtained using conventionaltechniques known to those with skill in the art, and the fragments areevaluated for function in the same manner as are intact binding proteinssuch as, for example, antibodies.

The term “neutralizing” refers to a binding protein or antigen-bindingfragment thereof (for example, an antibody) that reduces or blocks theactivity of a signaling pathway or an antigen, e.g., IL-21/IL-21Rsignaling pathway or IL-21R antigen. “An anti-product antibody,” as usedherein, refers to an antibody formed in response to exogenous protein,e.g., an anti-IL-21R antibody. “A neutralizing anti-product antibody,”as used herein, refers to an anti-product antibody that blocks the invivo activity of the exogenously introduced protein, e.g., ananti-IL-21R antibody. In some embodiments of the invention, aneutralizing anti-product antibody diminishes in vivo activity of anIL-21R antibody, e.g., in vivo pharmacodynamic (PD) activity of anIL-21R antibody (such as the ability of an anti-IL-21R antibody tomodulate expression of IL-21-responsive cytokines or genes).

The term “effective amount” refers to a dosage or amount that issufficient to regulate IL-21R activity to ameliorate or lessen theseverity of clinical symptoms or achieve a desired biological outcome,e.g., decreased T cell and/or B cell activity, suppression ofautoimmunity, suppression of transplant rejection.

The term “human binding protein” includes binding proteins havingvariable and constant regions corresponding substantially to humangermline immunoglobulin sequences known in the art, including, forexample, those described by Kabat et al. (5th ed. 1991) Sequences ofProteins of Immunological Interest, U.S. Department of Health and HumanServices, NIH Publication No. 91-3242. The human antibodies of theinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample, in the CDRs, and in particular, CDR3. The human antibody canhave at least one, two, three, four, five, or more positions replacedwith an amino acid residue that is not encoded by the human germlineimmunoglobulin sequence.

The phrases “inhibit,” “antagonize,” “block,” or “neutralize” IL-21Ractivity and its cognates refer to a reduction, inhibition, or otherwisediminution of at least one activity of IL-21R due to binding ananti-IL-21R antibody, wherein the reduction is relative to the activityof IL-21R in the absence of the same antibody. The IL-21R activity canbe measured using any technique known in the art Inhibition orantagonism does not necessarily indicate a total elimination of theIL-21R biological activity. A reduction in activity may be about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

The terms “interleukin-21 receptor” or “IL-21R” or the like refer to aclass I cytokine family receptor, also known as MU-1 (see, e.g., U.S.patent application Ser. No. 09/569,384 and U.S. Application PublicationNos. 2004/0265960; 2006/0159655; 2006/0024268; and 2008/0241098), NILRor zalpha11 (see, e.g., International Application Publication No. WO01/085792; Parrish-Novak et al. (2000) supra; Ozaki et al. (2000)supra), that binds to an IL-21 ligand. IL-21R is homologous to theshared p chain of the IL-2 and IL-15 receptors, and IL-4α (Ozaki et al.(2000) supra). Upon ligand binding, IL-21R is capable of interactingwith a common gamma cytokine receptor chain (γc) and inducing thephosphorylation of STAT1 and STAT3 (Asao et al. (2001) supra) or STATS(Ozaki et al. (2000) supra). IL-21R shows widespread lymphoid tissuedistribution. The terms “interleukin-21 receptor” or “IL-21R” or thelike also refer to a polypeptide (preferably of mammalian origin, e.g.,murine or human IL-21R) or, as context requires, a polynucleotideencoding such a polypeptide, that is capable of interacting with IL-21(preferably IL-21 of mammalian origin, e.g., murine or human IL-21) andhas at least one of the following features: (1) an amino acid sequenceof a naturally occurring mammalian IL-21R polypeptide or a fragmentthereof, e.g., an amino acid sequence set forth in SEQ ID NO:2(human—corresponding to GENBANK® (U.S. Dept. of Health and HumanServices, Bethesda, Md.) Accession No. NP_(—)068570) or SEQ ID NO:4(murine—corresponding to GENBANK® Acc. No. NP_(—)068687), or a fragmentthereof; (2) an amino acid sequence substantially homologous to, e.g.,at least 85%, 90%, 95%, 98%, or 99% homologous to, an amino acidsequence set forth in SEQ ID NO:2 or SEQ ID NO:4, or a fragment thereof;(3) an amino acid sequence that is encoded by a naturally occurringmammalian IL-21R nucleotide sequence or fragment thereof (e.g., SEQ IDNO:1 (human—which comprises an open reading frame corresponding to theopen reading frame of GENBANK® Accession No. NM_(—)021798) or SEQ IDNO:3 (murine—which comprises an open reading frame corresponding to theopen reading frame of GENBANK® Acc. No. NM_(—)021887), or a fragmentthereof); (4) an amino acid sequence encoded by a nucleotide sequencethat is substantially homologous to, e.g., at least 85%, 90%, 95%, 98%,or 99% homologous to, a nucleotide sequence set forth in SEQ ID NO:1 orSEQ ID NO:3 or a fragment thereof; (5) an amino acid sequence encoded bya nucleotide sequence degenerate to a naturally occurring IL-21Rnucleotide sequence or a fragment thereof, e.g., SEQ ID NO:1 or SEQ IDNO:3, or a fragment thereof; or (6) a nucleotide sequence thathybridizes to one of the foregoing nucleotide sequences under stringentconditions, e.g., highly stringent conditions. In addition, othernonhuman and nonmammalian IL-21Rs are contemplated as useful in thedisclosed methods.

The term “interleukin-21” or “IL-21” refers to a cytokine that showssequence homology to IL-2, IL-4 and IL-15 (Parrish-Novak et al. (2000)supra), and binds to an IL-21R. Such cytokines share a common fold intoa “four-helix-bundle” structure that is representative of the family.IL-21 is expressed primarily in activated CD4⁺ T cells, and has beenreported to have effects on NK, B and T cells (Parrish-Novak et al.(2000) supra; Kasaian et al. (2002) supra). Upon IL-21 binding toIL-21R, activation of IL-21R leads to, e.g., STATS or STAT3 signaling(Ozaki et al. (2000) supra). The term “interleukin-21” or “IL-21” alsorefers to a polypeptide (preferably of mammalian origin, e.g., murine orhuman IL-21), or as context requires, a polynucleotide encoding such apolypeptide, that is capable of interacting with IL-21R (preferably ofmammalian origin, e.g., murine or human IL-21R) and has at least one ofthe following features: (1) an amino acid sequence of a naturallyoccurring mammalian IL-21 or a fragment thereof, e.g., an amino acidsequence set forth in SEQ ID NO:212 (human), or a fragment thereof; (2)an amino acid sequence substantially homologous to, e.g., at least 85%,90%, 95%, 98%, or 99% homologous to, an amino acid sequence set forth inSEQ ID NO:212, or a fragment thereof; (3) an amino acid sequence that isencoded by a naturally occurring mammalian IL-21 nucleotide sequence ora fragment thereof (e.g., SEQ ID NO:211 (human), or a fragment thereof);(4) an amino acid sequence encoded by a nucleotide sequence that issubstantially homologous to, e.g., at least 85%, 90%, 95%, 98%, or 99%homologous to, a nucleotide sequence set forth in SEQ ID NO:211 or afragment thereof; (5) an amino acid sequence encoded by a nucleotidesequence degenerate to a naturally occurring IL-21 nucleotide sequenceor a fragment thereof; or (6) a nucleotide sequence that hybridizes toone of the foregoing nucleotide sequences under stringent conditions,e.g., highly stringent conditions.

The terms “IL-21R activity” and the like (e.g., “activity of IL-21R,”“IL-21/IL-21R activity”) refer to at least one cellular processinitiated or interrupted as a result of IL-21R binding. IL-21Ractivities include, but are not limited to: (1) interacting with, e.g.,binding to, a ligand, e.g., an IL-21 polypeptide; (2) associating withor activating signal transduction (also called “signaling,” which refersto the intracellular cascade occurring in response to a particularstimuli) and signal transduction molecules (e.g., gamma chain (γc) andJAK1), and/or stimulating the phosphorylation and/or activation of STATproteins, e.g., STATS and/or STAT3; (3) modulating the proliferation,differentiation, effector cell function, cytolytic activity, cytokinesecretion, and/or survival of immune cells, e.g., T cells, NK cells, Bcells, macrophages, regulatory T cells (Tregs) and megakaryocytes; and(4) modulating expression of IL-21-responsive genes or cytokines, e.g.,modulating IL-21 effects on the level of expression of, e.g., TNF, IFNγ,IL-6, IL-8, IL-10, CD19, STAT3, ICAM-1, TBX21, CSF1, GZMB, PRF1, IL-2Rα,IL-21R, etc.

As used herein, “in vitro-generated antibody” refers to an antibodywhere all or part of the variable region (e.g., at least one CDR) isgenerated in a nonimmune cell selection (e.g., an in vitro phagedisplay, protein chip, or any other method in which candidate sequencescan be tested for their ability to bind to an antigen).

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it was derived. The term also refers topreparations where the isolated protein is sufficiently pure forpharmaceutical compositions, or is at least 70-80% (w/w) pure, at least80-90% (w/w) pure, at least 90-95% (w/w) pure, or at least 95%, 96%,97%, 98%, 99%, or 100% (w/w) pure.

The phrase “percent identical” or “percent identity” refers to thesimilarity between at least two different sequences. This percentidentity can be determined by standard alignment algorithms, forexample, the Basic Local Alignment Search Tool (BLAST) described byAltshul et al. ((1990) J. Mol. Biol. 215:403-10); the algorithm ofNeedleman et al. ((1970) J. Mol. Biol. 48:444-53); or the algorithm ofMeyers et al. ((1988) Comput. Appl. Biosci. 4:11-17). A set ofparameters may be the Blosum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5. Thepercent identity between two amino acid or nucleotide sequences can alsobe determined using the algorithm of Meyers and Miller ((1989) CABIOS4:11-17), which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4. The percent identity is usually calculated bycomparing sequences of similar length.

The term “repertoire” refers to at least one nucleotide sequence derivedwholly or partially from at least one sequence encoding at least oneimmunoglobulin. The sequence(s) may be generated by rearrangement invivo of the V, D, and J segments of heavy chains, and the V and Jsegments of light chains. Alternatively, the sequence(s) can begenerated from a cell in response to which rearrangement occurs, e.g.,in vitro stimulation. Alternatively, part or all of the sequence(s) maybe obtained by DNA splicing, nucleotide synthesis, mutagenesis, or othermethods (see, e.g., U.S. Pat. No. 5,565,332). A repertoire may includeonly one sequence or may include a plurality of sequences, includingones in a genetically diverse collection.

The terms “specific binding,” “specifically binds,” and the like referto two molecules forming a complex that is relatively stable underphysiologic conditions. Specific binding is characterized by a highaffinity and a low-to-moderate capacity as distinguished fromnonspecific binding, which usually has a low affinity with amoderate-to-high capacity. Typically, binding is considered specificwhen the association constant Ka is higher than about 10⁶M⁻¹s⁻¹. Ifnecessary, nonspecific binding can be reduced without substantiallyaffecting specific binding by varying the binding conditions. Theappropriate binding conditions, such as concentration of bindingprotein, ionic strength of the solution, temperature, time allowed forbinding, concentration of a blocking agent (e.g., serum albumin or milkcasein), etc., can be improved by a skilled artisan using routinetechniques. Illustrative conditions are set forth herein, but otherconditions known to the person of ordinary skill in the art fall withinthe scope of this invention.

As used herein, the terms “stringent,” “stringency,” and the likedescribe conditions for hybridization and washing. The isolatedpolynucleotides of the present invention can be used as hybridizationprobes and primers to identify and isolate nucleic acids havingsequences identical to or similar to those encoding the disclosedpolynucleotides. Therefore, polynucleotides isolated in this fashion maybe used to produce binding proteins against IL-21R or to identify cellsexpressing such binding proteins. Hybridization methods for identifyingand isolating nucleic acids include polymerase chain reaction (PCR),Southern hybridizations, in situ hybridization and Northernhybridization, and are well known to those skilled in the art.

Hybridization reactions can be performed under conditions of differentstringencies. The stringency of a hybridization reaction includes thedifficulty with which any two nucleic acid molecules will hybridize toone another and the conditions under which they will remain hybridized.Preferably, each hybridizing polynucleotide hybridizes to itscorresponding polynucleotide under reduced stringency conditions, morepreferably stringent conditions, and most preferably highly stringentconditions. Stringent conditions are known to those skilled in the artand can be found in, e.g., Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989) 6.3.1-6.3.6. Both aqueous and nonaqueousmethods are described in this reference, and either can be used. Oneexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by atleast one wash in 0.2×SSC/0.1% SDS at 50° C. Stringent hybridizationconditions are also accomplished with wash(es) in, e.g., 0.2×SSC/0.1%SDS at 55° C., 60° C., or 65° C. Highly stringent conditions include,e.g., hybridization in 0.5M sodium phosphate/7% SDS at 65° C., followedby at least one wash at 0.2×SSC/1% SDS at 65° C. Further examples ofstringency conditions are shown in Table 1 below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R.

TABLE 1 Hybridization Conditions Hybrid Hybridization Wash LengthTemperature Temperature Condition Hybrid (bp)¹ and Buffer² and Buffer² ADNA:DNA >50 65° C.; 1X SSC -or- 65° C.; 0.3X SSC 42° C.; 1X SSC, 50%formamide B DNA:DNA <50 T_(B)*; 1X SSC T_(B)*; 1X SSC C DNA:RNA >50 67°C.; 1X SSC -or- 67° C.; 0.3X SSC 45° C.; 1X SSC, 50% formamide D DNA:RNA<50 T_(D)*; 1X SSC T_(D)*; 1X SSC E RNA:RNA >50 70° C.; 1X SSC -or- 70°C.; 0.3X SSC 50° C.; 1X SSC, 50% formamide F RNA:RNA <50 T_(F)*; 1X SSCT_(F)*; 1X SSC G DNA:DNA >50 65° C.; 4X SSC -or- 65° C.; 1X SSC 42° C.;4X SSC, 50% formamide H DNA:DNA <50 T_(H)*; 4X SSC T_(H)*; 4X SSC IDNA:RNA >50 67° C.; 4X SSC -or- 67° C.; 1X SSC 45° C.; 4X SSC, 50%formamide J DNA:RNA <50 T_(J)*; 4X SSC T_(J)*; 4X SSC K RNA:RNA >50 70°C.; 4X SSC -or- 67° C.; 1X SSC 50° C.; 4X SSC, 50% formamide L RNA:RNA<50 T_(L)*; 2X SSC T_(L)*; 2X SSC M DNA:DNA >50 50° C.; 4X SSC -or- 50°C.; 2X SSC 40° C.; 6X SSC, 50% formamide N DNA:DNA <50 T_(N)*; 6X SSCT_(N)*; 6X SSC O DNA:RNA >50 55° C.; 4X SSC -or- 55° C.; 2X SSC 42° C.;6X SSC, 50% formamide P DNA:RNA <50 T_(P)*; 6X SSC T_(P)*; 6X SSC QRNA:RNA >50 60° C.; 4X SSC -or- 60° C.; 2X SSC 45° C.; 6X SSC, 50%formamide R RNA:RNA <50 T_(R)*; 4X SSC T_(R)*; 4X SSC ¹The hybrid lengthis that anticipated for the hybridized region(s) of the hybridizingpolynucleotides. When hybridizing a polynucleotide to a targetpolynucleotide of unknown sequence, the hybrid length is assumed to bethat of the hybridizing polynucleotide. When polynucleotides of knownsequence are hybridized, the hybrid length can be determined by aligningthe sequences of the polynucleotides and identifying the region orregions of optimal sequence complementarity. ²SSPE (1xSSPE is 0.15MNaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substituted forSSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridizationand wash buffers; washes are performed for 15 min after hybridization iscomplete. TB*-TR*: The hybridization temperature for hybrids anticipatedto be less than 50 base pairs in length should be 5-10° C. less than themelting temperature (T_(m)) of the hybrid, where T_(m) is determinedaccording to the following equations. For hybrids less than 18 basepairs in length, T_(m)(° C.) = 2(# of A + T bases) + 4(# of G + Cbases). For hybrids between 18 and 49 base pairs in length, T_(m)(° C.)= 81.5 + 16.6(log₁₀Na⁺) + 0.41(% G + C) − (600/N), where N is the numberof bases in the hybrid, and Na⁺ is the concentration of sodium ions inthe hybridization buffer (Na⁺ for 1X SSC = 0.165M). Additional examplesof stringency conditions for polynucleotide hybridization are providedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Chs. 9& 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, andAusubel et al. eds. (1995) Current Protocols in Molecular Biology Sects.2.10 & 6.3-6.4, John Wiley & Sons, Inc., herein incorporated byreference.

The isolated polynucleotides of the present invention may be used ashybridization probes and primers to identify and isolate DNAs havingsequences encoding allelic variants of the disclosed polynucleotides.Allelic variants are naturally occurring alternative forms of thedisclosed polynucleotides that encode polypeptides that are identical toor have significant similarity to the polypeptides encoded by thedisclosed polynucleotides. Preferably, allelic variants have at leastabout 90% sequence identity (more preferably, at least about 95%identity; most preferably, at least about 99% identity) with thedisclosed polynucleotides. The isolated polynucleotides of the presentinvention may also be used as hybridization probes and primers toidentify and isolate DNAs having sequences encoding polypeptideshomologous to the disclosed polynucleotides. These homologs arepolynucleotides and polypeptides isolated from a different species thanthat of the disclosed polypeptides and polynucleotides, or within thesame species, but with significant sequence similarity to the disclosedpolynucleotides and polypeptides. Preferably, polynucleotide homologshave at least about 50% sequence identity (more preferably, at leastabout 75% identity; most preferably, at least about 90% identity) withthe disclosed polynucleotides, whereas polypeptide homologs have atleast about 30% sequence identity (more preferably, at least about 45%identity; most preferably, at least about 60% identity) with thedisclosed binding proteins/polypeptides. Preferably, homologs of thedisclosed polynucleotides and polypeptides are those isolated frommammalian species. The isolated polynucleotides of the present inventionmay additionally be used as hybridization probes and primers to identifycells and tissues that express the binding proteins of the presentinvention and the conditions under which they are expressed.

The phrases “substantially as set out,” “substantially identical,” and“substantially homologous” mean that the relevant amino acid ornucleotide sequence (e.g., CDR(s), V_(H), or V_(L) domain(s)) will beidentical to or have insubstantial differences (e.g., through conservedamino acid substitutions) in comparison to the sequences which are setout. Insubstantial differences include minor amino acid changes, such asone or two substitutions in a five amino acid sequence of a specifiedregion. In the case of antibodies, the second antibody has the samespecificity and has at least about 50% of the affinity of the firstantibody.

Sequences substantially identical or homologous to the sequencesdisclosed herein are also part of this application. In some embodiments,the sequence identity can be about 85%, 90%, 95%, 96%, 97%, 98%, 99%, orhigher. Alternatively, substantial identity or homology exists when thenucleic acid segments will hybridize under selective hybridizationconditions (e.g., highly stringent hybridization conditions), to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form.

The term “therapeutic agent” or the like is a substance that treats orassists in treating a medical disorder or symptoms thereof. Therapeuticagents may include, but are not limited to, substances that modulateimmune cells or immune responses in a manner that complements the use ofanti-IL-21R binding proteins. In one embodiment of the invention, atherapeutic agent is a therapeutic antibody, e.g., an anti-IL-21Rantibody. In another embodiment of the invention, a therapeutic agent isa therapeutic binding protein, e.g., an anti-IL-21R nanobody.Nonlimiting examples and uses of therapeutic agents are describedherein.

As used herein, a “therapeutically effective amount” of an anti-IL-21Rbinding protein (e.g., an antibody) refers to an amount of the bindingprotein that is effective, upon single or multiple dose administrationto a subject (such as a human patient) for treating, preventing, curing,delaying, reducing the severity of, and/or ameliorating at least onesymptom of a disorder or a recurring disorder, or prolonging thesurvival of the subject beyond that expected in the absence of suchtreatment. In one embodiment, a therapeutically effective amount may bean amount of an anti-IL-21R binding protein that is sufficient tomodulate expression of at least one IL-21-responsive cytokine or gene.

The term “treatment” refers to a therapeutic or preventative measure.The treatment may be administered to a subject having a medical disorderor who ultimately may acquire the disorder, in order to prevent, cure,delay, reduce the severity of, and/or ameliorate one or more symptoms ofa disorder or a recurring disorder, or in order to prolong the survivalof a subject beyond that expected in the absence of such treatment.

Anti-IL-21R Binding Proteins

The disclosure of the present application provides novel anti-IL-21Rbinding proteins that comprise novel antigen-binding fragments. Numerousmethods known to those skilled in the art are available for obtainingbinding proteins or antigen-binding fragments thereof. For example,anti-IL-21R binding proteins that comprise antibodies can be producedusing recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).Monoclonal antibodies may also be produced by generation of hybridomasin accordance with known methods (see, e.g., Kohler and Milstein (1975)Nature 256:495-99). Hybridomas formed in this manner are then screenedusing standard methods, such as enzyme-linked immunosorbent assays(ELISA) and surface plasmon resonance (BIACORE™) analysis, to identifyone or more hybridomas that produce an antibody that specifically bindswith a particular antigen. Any form of the specified antigen may be usedas the immunogen, e.g., recombinant antigen, naturally occurring forms,any variants or fragments thereof, and antigenic peptides thereof.

One exemplary method of making binding proteins that comprise antibodiesincludes screening protein expression libraries, e.g., phage or ribosomedisplay libraries. Phage display is described, for example, in U.S. Pat.No. 5,223,409; Smith (1985) Science 228:1315-17; Clackson et al. (1991)Nature 352:624-28; Marks et al. (1991) J. Mol. Biol. 222:581-97; andInternational Application Publication Nos. WO 92/018619; WO 91/017271;WO 92/020791; WO 92/015679; WO 93/001288; WO 92/001047; WO 92/009690;and WO 90/002809.

In addition to the use of display libraries, the specified antigen canbe used to immunize a nonhuman animal, e.g., a cynomolgus monkey, achicken, or a rodent (e.g., a mouse, hamster, or rat). In oneembodiment, the nonhuman animal includes at least a part of a humanimmunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal binding proteins, such as antibodies, derived from the geneswith the desired specificity may be produced and selected (see, e.g.,XENOMOUSE™ (Amgen, Inc., Thousand Oaks, Calif.); Green et al. (1994)Nat. Genet. 7:13-21; U.S. Pat. No. 7,064,244; and InternationalApplication Publication Nos. WO 96/034096 and WO 96/033735).

In one embodiment of the invention, the binding proteins is a monoclonalantibody that is obtained from a nonhuman animal, and then modified(e.g., humanized, deimmunized, or chimeric) using recombinant DNAtechniques known in the art. A variety of approaches for making chimericantibodies have been described (see, e.g., Morrison et al. (1985) Proc.Natl. Acad. Sci. USA 81(21):6851-55; Takeda et al. (1985) Nature314(6010):452-54; U.S. Pat. Nos. 4,816,567 and 4,816,397; EuropeanApplication Publication Nos. EP 0 171 496 and EP 0 173 494; and UnitedKingdom Patent No. GB 2 177 096). Humanized binding proteins may also beproduced, for example, using transgenic mice that express human heavyand light chain genes, but are incapable of expressing the endogenousmouse immunoglobulin heavy and light chain genes. Winter (U.S. Pat. No.5,225,539) describes an exemplary CDR-grafting method that may be usedto prepare the humanized binding proteins described herein. All of theCDRs of a particular human binding protein may be replaced with at leasta portion of a nonhuman CDR, or only some of the CDRs may be replacedwith nonhuman CDRs. It is only necessary to replace the number of CDRsrequired for binding of the humanized binding protein to a predeterminedantigen.

Humanized binding proteins or fragments thereof can be generated byreplacing sequences of the Fv variable domain that are not directlyinvolved in antigen binding with equivalent sequences from human Fvvariable domains. Exemplary methods for generating humanized bindingproteins or fragments thereof are provided by, e.g., Morrison (1985)Science 229:1202-07; Oi et al. (1986) BioTechniques 4:214; and U.S. Pat.Nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213. Thosemethods include isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable domainsfrom at least one of a heavy or light chain. Such nucleic acids may beobtained from a hybridoma producing an antibody against a predeterminedtarget, as described above, as well as from other sources. Therecombinant DNA encoding the humanized antibody molecule can then becloned into an appropriate expression vector.

In certain embodiments, a humanized binding protein is improved by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or backmutations. Such alteredimmunoglobulin molecules can be made by any of several techniques knownin the art, (see, e.g., Teng et al. (1983) Proc. Natl. Acad. Sci. USA80:7308-73; Kozbor et al. (1983) Immunol. Today 4:7279; Olsson et al.(1982) Meth. Enzymol. 92:3-16); International Application PublicationNo. WO 92/006193; and European Patent No. EP 0 239 400).

A binding protein or fragment thereof may also be modified by specificdeletion of human T cell epitopes or “deimmunization” by the methodsdisclosed in, e.g., International Application Publication Nos. WO98/052976 and WO 00/034317. Briefly, the heavy and light chain variabledomains of a binding protein (such as, for example, a binding proteinderived from an antibody) can be analyzed for peptides that bind to MHCClass II; these peptides represent potential T cell epitopes (as definedin, e.g., International Application Publication Nos. WO 98/052976 and WO00/034317). For detection of potential T cell epitopes, a computermodeling approach termed “peptide threading” can be applied, and inaddition a database of human MHC class II binding peptides can besearched for motifs present in the V_(H) and V_(L) sequences, asdescribed in International Application Publication Nos. WO 98/052976 andWO 00/034317. These motifs bind to any of the 18 major MHC Class II DRallotypes and thus, constitute potential T cell epitopes. Potential Tcell epitopes detected can be eliminated by substituting small numbersof amino acid residues in the variable domains or by single amino acidsubstitutions. Typically, conservative substitutions are made. Often,but not exclusively, an amino acid common to a position in humangermline antibody sequences may be used. Human germline sequences aredisclosed in, e.g., Tomlinson et al. (1992) J. Mol. Biol. 227:776-98;Cook et al. (1995) Immunol. Today 16(5):237-42; Chothia et al. (1992) J.Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-38.The V BASE directory provides a comprehensive directory of humanimmunoglobulin variable region sequences (compiled by Tomlinson et al.,MRC Centre for Protein Engineering, Cambridge, UK). These sequences canbe used as a source of human sequence, e.g., for framework regions andCDRs. Consensus human framework regions can also be used, as describedin, e.g., U.S. Pat. No. 6,300,064.

In certain embodiments, a binding protein can contain an alteredimmunoglobulin constant or Fc region. For example, binding proteinsproduced in accordance with the teachings herein may bind more stronglyor with more specificity to effector molecules such as complement and/orFc receptors, which can control several immune functions of the bindingprotein such as effector cell activity, lysis, complement-mediatedactivity, binding protein clearance, and binding protein half-life.Typical Fc receptors that bind to an Fc region of a binding protein(e.g., an IgG antibody) include, but are not limited to, receptors ofthe FcγRI, FcγRII, and FcRn subclasses, including allelic variants andalternatively spliced forms of these receptors. Fc receptors arereviewed in, e.g., Ravetch and Kinet (1991) Annu. Rev. Immunol.9:457-92; Capel et al. (1994) Immunomethods 4:25-34; and de Haas et al.(1995) J. Lab. Clin. Med. 126:330-41. For additional bindingprotein/antibody production techniques, see, e.g., Antibodies: ALaboratory Manual (1988) Harlow et al. eds., Cold Spring HarborLaboratory. The present invention is not necessarily limited to anyparticular source, method of production, or other special characteristicof a binding protein or an antibody.

Binding proteins comprising antibodies (immunoglobulins) are typicallytetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each and two heavy (H) chains of approximately 50kDa each. Two types of light chains, termed lambda (λ) and kappa (κ),may be found in antibodies. Depending on the amino acid sequence of theconstant domain of heavy chains, immunoglobulins can be assigned to fivemajor classes: A, D, E, G, and M, and several of these may be furtherdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1,and IgA2. Each light chain includes an N-terminal variable (V) domain(V_(L)) and a constant (C) domain (C_(L)). Each heavy chain includes anN-terminal V domain (V_(H)), three or four C domains (C_(H)s), and ahinge region. The C_(H) domain most proximal to V_(H) is designated asC_(H)1. The V_(H) and V_(L) domains consist of four regions ofrelatively conserved sequences called framework regions (FR1, FR2, FR3,and FR4) that form a scaffold for three regions of hypervariablesequences, called CDRs. The CDRs contain most of the residuesresponsible for specific interactions of the antibody with the antigen.CDRs are referred to as CDR1, CDR2, and CDR3. CDR constituents on theheavy chain are referred to as H1, H2, and H3 (also referred to hereinas CDR H1, CDR H2, and CDR H3, respectively), while CDR constituents onthe light chain are referred to as L1, L2, and L3 (also referred toherein as CDR L1, CDR L2, and CDR L3, respectively).

CDR3 is typically the greatest source of molecular diversity within theantigen-binding site. CDR H3, for example, can be as short as two aminoacid residues or greater than 26 amino acids. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known in the art. For a review of antibody structure, see,e.g., Harlow et al. (1988) supra. One of skill in the art will recognizethat each subunit structure, e.g., a C_(H), V_(H), C_(L), V_(L), CDR,and/or FR structure, comprises active fragments, e.g., the portion ofthe V_(H), V_(L), or CDR subunit that binds to the antigen, i.e., theantigen-binding fragment, or, e.g., the portion of the C_(H) subunitthat binds to and/or activates, e.g., an Fc receptor and/or complement.The CDRs typically refer to the Kabat CDRs (as described in Kabat et al.(1991) supra). Another standard for characterizing the antigen-bindingsite is to refer to the hypervariable loops as described in, e.g.,Chothia et al. (1992) supra and Tomlinson et al. (1995) supra. Stillanother standard is the “AbM” definition used by Oxford Molecular's AbMantibody modeling software (see, generally, e.g., Protein Sequence andStructure Analysis of Antibody Variable Domains in: Antibody Engineering(2001) Duebel and Kontermann eds., Springer-Verlag, Heidelberg).Embodiments described with respect to Kabat CDRs can alternatively beimplemented using similar described relationships with respect toChothia hypervariable loops or to the AbM-defined loops.

The Fab fragment consists of V_(H)-C_(H)1 and V_(L)-C_(L) domainscovalently linked by a disulfide bond between the constant regions. TheF_(v) fragment is smaller and consists of V_(H) and V_(L) domainsnoncovalently linked. To overcome the tendency of noncovalently linkeddomains to dissociate, an scFv can be constructed. The scFv contains aflexible polypeptide that links (1) the C-terminus of V_(H) to theN-terminus of V_(L), or (2) the C-terminus of V_(L) to the N-terminus ofV_(H). A 15-mer (Gly₄Ser)₃ peptide, for example, may be used as alinker, but other linkers are known in the art.

The sequence of antibody genes after assembly and somatic mutation ishighly varied, and these varied genes are estimated to encode 10¹⁰different antibody molecules (Immunoglobulin Genes (2nd ed. 1995) Jonioet al. eds., Academic Press, San Diego, Calif.).

In certain embodiments of the invention, the binding protein is a singledomain binding protein. Single domain binding proteins include bindingproteins wherein the CDRs are part of a single domain polypeptide.Examples include, but are not limited to, heavy chain binding proteins,binding proteins that are naturally devoid of light chains, singledomain binding proteins derived from conventional four-chain antibodies,engineered binding proteins, and single domain protein scaffolds otherthan those derived from antibodies. Single domain binding proteinsinclude any known in the art, as well as any future-determined or-learned single domain binding proteins.

Single domain binding proteins may be derived from any speciesincluding, but not limited to, mouse, human, camel, llama, fish, shark,goat, rabbit, chicken, and bovine. In one aspect of the invention, thesingle domain binding protein can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainbinding proteins derived from a variable region of NAR (IgNARs) aredescribed in, e.g., International Application Publication No. WO03/014161 and Streltsov (2005) Protein Sci. 14:2901-09. Single domainbinding proteins also include naturally occurring single domain bindingproteins known in the art as heavy chain antibodies devoid of lightchains. This variable domain derived from a heavy chain antibodynaturally devoid of a light chain is known herein as a VHH, or ananobody, to distinguish it from the conventional V_(H) of four-chainimmunoglobulins. Such a VHH molecule can be derived from antibodiesraised in Camelidae species, for example, in camel, llama, dromedary,alpaca, and guanaco, and is sometimes called a camelid or camelizedvariable domain (see, e.g., Muyldermans (2001) J. Biotechnol.74(4):277-302, incorporated herein by reference). Other species besidesthose in the family Camelidae may also produce heavy chain bindingproteins naturally devoid of light chains. VHH molecules are about tentimes smaller than IgG molecules. They are single polypeptides and arevery stable, resisting extreme pH and temperature conditions. Moreover,they are resistant to the actions of proteases, which is not the casefor conventional antibodies. Furthermore, in vitro expression of VHHscan produce high-yield, properly folded functional VHHs. In addition,binding proteins generated in camelids will recognize epitopes otherthan those recognized by antibodies generated in vitro via antibodylibraries or via immunization of mammals other than camelids (see, e.g.,International Application Publication Nos. WO 97/049805 and WO94/004678, which are incorporated herein by reference).

A “bispecific” or “bifunctional” binding protein is an artificial hybridbinding protein having two different heavy/light chain pairs and twodifferent binding sites. Bispecific binding proteins can be produced bya variety of methods including fusion of hybridomas or linking of Fab′fragments (see, e.g., Songsivilai and Lachmann (1990) Clin. Exp.Immunol. 79:315-21; Kostelny et al. (1992) J. Immunol. 148:1547-53). Inone embodiment, the bispecific binding protein comprises a first bindingdomain polypeptide, such as a Fab′ fragment, linked via animmunoglobulin constant region to a second binding domain polypeptide.

Another binding protein according to the invention can comprise, forexample, a binding domain-immunoglobulin fusion protein that includes abinding domain polypeptide that is fused or otherwise connected to animmunoglobulin hinge or hinge-acting region polypeptide, which in turnis fused or otherwise connected to a region comprising one or morenative or engineered constant regions from an immunoglobulin heavychain, other than C_(H)1, for example, the C_(H)2 and C_(H)3 regions ofIgG and IgA1 or the C_(H)3 and C_(H)4 regions of IgE (see, e.g., U.S.Application Publication No. 2005/0136049, which is incorporated byreference herein, for a more complete description). The bindingdomain-immunoglobulin fusion protein can further include a region thatincludes a native or engineered immunoglobulin heavy chain C_(H)2constant region polypeptide (or C_(H)3 in the case of a constructderived in whole or in part from IgE) that is fused or otherwiseconnected to the hinge region polypeptide and a native or engineeredimmunoglobulin heavy chain C_(H)3 constant region polypeptide (or C_(H)4in the case of a construct derived in whole or in part from IgE) that isfused or otherwise connected to the C_(H)2 constant region polypeptide(or C_(H)3 in the case of a construct derived in whole or in part fromIgE). Typically, such binding domain-immunoglobulin fusion proteins arecapable of at least one immunological activity selected from the groupconsisting of antibody-dependent cell-mediated cytotoxicity (ADCC),complement fixation, and/or binding to a target, for example, a targetantigen, such as human IL-21R.

Binding proteins of the invention can also comprise peptide mimetics.Peptide mimetics are peptide-containing molecules that mimic elements ofprotein secondary structure (see, for example, Johnson et al. PeptideTurn Mimetics in Biotechnology and Pharmacy (1993), Pezzuto et al. eds.,Chapman and Hall, New York, incorporated by reference herein in itsentirety). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those between antibody and antigen. A peptide mimetic isexpected to permit molecular interactions similar to the naturalmolecule. These principles may be used to engineer second generationmolecules having many of the natural properties of the targetingpeptides disclosed herein, but with altered and potentially improvedcharacteristics.

Other embodiments of binding proteins useful for practicing theinvention include fusion proteins. These molecules generally have all ora substantial portion of a targeting peptide, for example, IL-21R or ananti IL-21R antibody, linked at the N- or C-terminus, to all or aportion of a second polypeptide or protein. For example, fusion proteinsmay employ leader (or signal) sequences from other species to permit therecombinant expression of a protein in a heterologous host. For example,amino acid sequences, or nucleic acid sequences encoding amino acidsequences, of the binding proteins and antigen-binding fragments thereofof the present invention comprising leader (or signal) sequence may beselected from SEQ ID NOs:87-109 and 239-248. Another useful fusionincludes the addition of an immunologically active domain, such as abinding protein epitope, to facilitate purification of the fusionprotein. Inclusion of a cleavage site at or near the fusion junctionwill facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include the linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals, or transmembrane regions. Examples ofproteins or peptides that may be incorporated into a fusion proteininclude, but are not limited to, cytostatic proteins, cytocidalproteins, pro-apoptosis agents, anti-angiogenic agents, hormones,cytokines, growth factors, peptide drugs, antibodies, Fab fragments ofantibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins,cell adhesion proteins, and binding proteins. Methods of generatingfusion proteins are well known to those of skill in the art. Suchproteins can be produced, for example, by chemical attachment usingbifunctional cross-linking reagents, by de novo synthesis of thecomplete fusion protein, or by attachment of a DNA sequence encoding thetargeting peptide to a DNA sequence encoding the second peptide orprotein, followed by expression of the intact fusion protein.

In one embodiment, the targeting peptide, for example, IL-21R, is fusedwith an immunoglobulin heavy chain constant region, such as an Fcfragment, which contains two constant region domains and a hinge region,but lacks the variable region (see, e.g., U.S. Pat. Nos. 6,018,026 and5,750,375, incorporated by reference herein). The Fc region may be anaturally occurring Fc region, or may be altered to improve certainqualities, e.g., therapeutic qualities, circulation time, reducedaggregation. Peptides and proteins fused to an Fc region typicallyexhibit a greater half-life in vivo than the unfused counterpart does.In addition, a fusion to an Fc region permitsdimerization/multimerization of the fusion polypeptide.

One aspect of the present invention comprises binding proteins andantigen-binding fragments thereof that bind IL-21R. The disclosureprovides novel CDRs that have been derived from human immunoglobulingene libraries. The protein structure that is generally used to carry aCDR is an antibody heavy or light chain or a portion thereof, whereinthe CDR is localized to a region associated with a naturally occurringCDR. The structures and locations of variable domains may be determinedas described in Kabat et al. ((1991) supra).

Illustrative embodiments of the binding proteins (and antigen-bindingfragments thereof) of the invention are identified as AbA-AbU, H3-H6,L1-L6, L8-L21, and L23-L25. DNA and amino acid sequences of thenonlimiting illustrative embodiments of the anti-IL-21R binding proteinsof the invention are set forth in SEQ ID NOs:5-195, 213-229, and239-248. DNA and amino acid sequences of some illustrative embodimentsof the anti-IL-21R binding proteins of the invention, including theirscFv fragments, V_(H) and V_(L) domains, and CDRs, as well as theirpresent codes and previous designations, are set forth in FIGS. 17-25,and Tables 2A and 2B.

TABLE 2A Correlation of Present Antibody Codes and Previous DesignationsPresent Code Previous Designation AbA VHP/VL2 AbB VHP/VL3 AbC VHP/VL11AbD VHP/VL13 AbE VHP/VL14 AbF VHP/VL17 AbG VHP/VL18 AbH VHP/VL19 AbIVHP/VL24 AbJ VH3/VLP AbK VH3/VL3 AbL VH3/VL13 AbM VH6/VL13 AbN VH6/VL24AbO VHP/VL16; VHPTM/VL16 AbP VHP/VL20; VHPTM/VL20 AbQ VH3/VL2; VH3DM/VL2AbR VH3/VL18; VH3DM/VL18 AbS VHP/VL6; VHPTM/VL6; VL6 AbT VHP/VL9;VHPTM/VL9; VL9 AbU VHP/VL25; VHPTM/VL25 AbV VH3TM/VL2 AbW VH3TM/VL18 AbXVHPDM/VL9 AbY VHPg4/VL9 AbZ VHPWT/VL9

TABLE 2B Amino Acid and Nucleotide Sequences of V_(H) and V_(L) Domains,scFv, and CDRs of Illustrative Binding Proteins of the Invention H3 H4H5 H6 L1 REGION TYPE SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 14NO: 16 NO: 18 NO: 20 NO: 6 V_(L) AA NO: 10 NO: 10 NO: 10 NO: 10 NO: 22scFv AA NO: 110 NO: 112 NO: 114 NO: 116 NO: 118 CDR H1 AA NO: 163 NO:163 NO: 163 NO: 163 NO: 163 CDR H2 AA NO: 164 NO: 164 NO: 164 NO: 164NO: 164 CDR H3 AA NO: 165 NO: 166 NO: 167 NO: 168 NO: 169 CDR L1 AA NO:194 NO: 194 NO: 194 NO: 194 NO: 194 CDR L2 AA NO: 195 NO: 195 NO: 195NO: 195 NO: 195 CDR L3 AA NO: 170 NO: 170 NO: 170 NO: 170 NO: 171 V_(H)DNA NO: 13 NO: 15 NO: 17 NO: 19 NO: 5 V_(L) DNA NO: 9 NO: 9 NO: 9 NO: 9NO: 21 scFv DNA NO: 109 NO: 111 NO: 113 NO: 115 NO: 117 L2 L3 L4 L5 L6REGION TYPE SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 6 NO: 6 NO:6 NO: 6 NO: 6 V_(L) AA NO: 24 NO: 26 NO: 28 NO: 30 NO: 32 scFv AA NO:120 NO: 122 NO: 124 NO: 126 NO: 128 CDR H1 AA NO: 163 NO: 163 NO: 163NO: 163 NO: 163 CDR H2 AA NO: 164 NO: 164 NO: 164 NO: 164 NO: 164 CDR H3AA NO: 169 NO: 169 NO: 169 NO: 169 NO: 169 CDR L1 AA NO: 194 NO: 194 NO:194 NO: 194 NO: 194 CDR L2 AA NO: 195 NO: 195 NO: 195 NO: 195 NO: 195CDR L3 AA NO: 172 NO: 173 NO: 174 NO: 175 NO: 176 V_(H) DNA NO: 5 NO: 5NO: 5 NO: 5 NO: 5 V_(L) DNA NO: 23 NO: 25 NO: 27 NO: 29 NO: 31 scFv DNANO: 119 NO: 121 NO: 123 NO: 125 NO: 127 L8 L9 L10 L11 L12 REGION TYPESEQ ID SEQ ID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 6 NO: 6 NO6 NO: 6 NO: 6V_(L) AA NO: 34 NO: 36 NO: 38 NO: 40 NO: 42 scFv AA NO: 130 NO: 132 NO:134 NO: 136 NO: 138 CDR H1 AA NO: 163 NO: 163 NO: 163 NO: 163 NO: 163CDR H2 AA NO: 164 NO: 164 NO: 164 NO: 164 NO: 164 CDR H3 AA NO: 169 NO:169 NO: 169 NO: 169 NO: 169 CDR L1 AA NO: 194 NO: 194 NO: 194 NO: 194NO: 194 CDR L2 AA NO: 195 NO: 195 NO: 195 NO: 195 NO: 195 CDR L3 AA NO:177 NO: 178 NO: 179 NO: 180 NO: 181 V_(H) DNA NO: 5 NO: 5 NO: 5 NO: 5NO: 5 V_(L) DNA NO: 33 NO: 35 NO: 37 NO: 39 NO: 41 scFv DNA NO: 129 NO:131 NO: 133 NO: 135 NO: 137 L13 L14 L15 L16 L17 REGION TYPE SEQ ID SEQID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 6 NO: 6 NO: 6 NO: 6 NO: 6 V_(L) AANO: 44 NO: 46 NO: 48 NO: 50 NO: 52 scFv AA NO: 140 NO: 142 NO: 144 NO:146 NO: 148 CDR H1 AA NO: 163 NO: 163 NO: 163 NO: 163 NO: 163 CDR H2 AANO: 164 NO: 164 NO: 164 NO: 164 NO: 164 CDR H3 AA NO: 169 NO: 169 NO:169 NO: 169 NO: 169 CDR L1 AA NO: 194 NO: 194 NO: 194 NO: 194 NO: 194CDR L2 AA NO: 195 NO: 195 NO: 195 NO: 195 NO: 195 CDR L3 AA NO: 182 NO:183 NO: 184 NO: 185 NO: 186 V_(H) DNA NO: 5 NO: 5 NO: 5 NO: 5 NO: 5V_(L) DNA NO: 43 NO: 45 NO: 47 NO: 49 NO: 51 scFv DNA NO: 139 NO: 141NO: 143 NO: 145 NO: 147 L18 L19 L20 L21 L23 REGION TYPE SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID V_(H) AA NO: 6 NO: 6 NO: 6 NO: 6 NO: 6 V_(L) AA NO:54 NO: 56 NO: 58 NO: 60 NO: 62 scFv AA NO: 150 NO: 152 NO: 154 NO: 156NO: 158 CDR H1 AA NO: 163 NO: 163 NO: 163 NO: 163 NO: 163 CDR H2 AA NO:164 NO: 164 NO: 164 NO: 164 NO: 164 CDR H3 AA NO: 169 NO: 169 NO: 169NO: 169 NO: 169 CDR L1 AA NO: 194 NO: 194 NO: 194 NO: 194 NO: 194 CDR L2AA NO: 195 NO: 195 NO: 195 NO: 195 NO: 195 CDR L3 AA NO: 187 NO: 188 NO:189 NO: 190 NO: 191 V_(H) DNA NO: 5 NO: 5 NO: 5 NO: 5 NO: 5 V_(L) DNANO: 53 NO: 55 NO: 57 NO: 59 NO: 61 scFv DNA NO: 149 NO: 151 NO: 153 NO:155 NO: 157 L24 L25 REGION TYPE SEQ ID SEQ ID V_(H) AA NO: 6 NO: 6 V_(L)AA NO: 64 NO: 66 scFv AA NO: 160 NO: 162 CDR H1 AA NO: 163 NO: 163 CDRH2 AA NO: 164 NO: 164 CDR H3 AA NO: 169 NO: 169 CDR L1 AA NO: 194 NO:194 CDR L2 AA NO: 195 NO: 195 CDR L3 AA NO: 192 NO: 193 V_(H) DNA NO: 5NO: 5 V_(L) DNA NO: 63 NO: 65 scFv DNA NO: 159 NO: 161

Anti-IL-21R binding proteins of the present invention may compriseantibody constant regions or parts thereof. For example, a V_(L) domainmay be attached at its C-terminal end to a light chain constant domainlike Cκ or Cλ. Similarly, a V_(H) domain, or portion thereof, may beattached to all or part of a heavy chain like IgA, IgD, IgE, IgG, andIgM, and any isotype subclass. Constant regions are known in the art(see, e.g., Kabat et al. (1991) supra). Therefore, binding proteinswithin the scope of this invention include V_(H) and V_(L) domains, orportions thereof, combined with constant regions known in the art.

Certain embodiments comprise a V_(H) domain, a V_(L) domain, or acombination thereof, of the Fv fragment from AbA-AbZ, H3-H6, L1-L6,L8-L21, and/or L23-L25. Further embodiments comprise one, two, three,four, five or six CDRs from the V_(H) and V_(L) domains. Bindingproteins whose CDR sequence(s) are the same as, or similar to (i.e.,differ insubstantially from), one or more CDR sequence(s) present withinthe sequences set forth in SEQ ID NOs:5-195, 213-229, and 239-248 areencompassed within the scope of the invention.

In certain embodiments, the V_(H) and/or V_(L) domains may be germlined,i.e., the FR of these domains are mutated using conventional molecularbiology techniques to match those produced by the germline cells. Inother embodiments, the FR sequences remain diverged from the consensusgermline sequences.

In one embodiment, mutagenesis is used to make a binding protein moresimilar to one or more germline sequences. This may be desirable whenmutations are introduced into the FR of a binding protein (e.g., anantibody) through somatic mutagenesis or through error prone PCR.Germline sequences for the V_(H) and V_(L) domains can be identified byperforming amino acid and nucleic acid sequence alignments against theVBASE database (MRC Center for Protein Engineering, UK). VBASE is acomprehensive directory of all human germline variable region sequencescompiled from over a thousand published sequences, including those inthe current releases of the GENBANK® and EMBL data libraries. In someembodiments, the FRs of scFvs are mutated in conformity with the closestmatches in the VBASE database and the CDR portions are kept intact.

In certain embodiments, binding proteins of the invention specificallyreact with an epitope that is the same as the epitope recognized byAbA-AbZ, H3-H6, L1-L6, L8-L21, or L23-L25, such that they competitivelyinhibit the binding of AbA-AbZ, H3-H6, L1-L6, L8-L21, or L23-L25 tohuman IL-21R. Such binding proteins can be determined in competitivebinding assays. In one embodiment, the association constant (K_(A)) ofthese binding proteins for human IL-21R is at least 10⁵ M⁻¹s⁻¹. Thebinding affinity may be determined using techniques known in the art,such as ELISA, biosensor technology (such as biospecific interactionanalysis) or other techniques, including those described in thisapplication.

It is contemplated that binding proteins of the invention may bind otherproteins, such as, for example, recombinant proteins comprising all or aportion of IL-21R.

One of ordinary skill in the art will recognize that the disclosedbinding proteins may be used to detect, measure, and/or inhibit proteinsthat differ somewhat from IL-21R. For example, these proteins may behomologs of IL-21R. Anti-IL-21R binding proteins are expected to bindproteins that comprise a sequence that is at least about 60%, 70%, 80%,90%, 95%, or more identical to any sequence of at least 100, 80, 60, 40,or 20 contiguous amino acids in the sequence set forth SEQ ID NOs:2 or4.

In addition to sequence homology analyses, epitope mapping (see, e.g.,Epitope Mapping Protocols (1996) Morris ed., Humana Press), andsecondary and tertiary structure analyses can be carried out to identifyspecific 3D structures assumed by the presently disclosed bindingproteins and their complexes with antigens. Such methods include, butare not limited to, x-ray crystallography (Engstom (1974) Biochem. Exp.Biol. 11:7-13) and computer modeling of virtual representations of thepresent binding proteins (Fletterick et al. (1986) Computer Graphics andMolecular Modeling, in Current Communications in Molecular Biology, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The disclosure provides a method for obtaining anti-IL-21R bindingproteins. The method comprises creating binding proteins with V_(H)and/or V_(L) sequence(s) that are altered from those sequences disclosedherein. Such binding proteins may be derived by a skilled artisan usingtechniques known in the art. For example, amino acid substitutions,deletions, or additions can be introduced in FR and/or CDR regions. FRchanges are usually designed to improve the stability and immunogenicityof the binding protein, while CDR changes are typically designed toincrease a binding protein's affinity for its antigen. The changes thatincrease affinity may be tested by altering one or more CDR sequencesand measuring the affinity of the binding protein for its target (see,e.g., Antibody Engineering (2nd ed. 1995) Borrebaeck ed., OxfordUniversity Press).

Binding proteins whose CDR sequences differ insubstantially from thoseset forth in or included within the sequences of SEQ ID NOs:5-195,213-229, and 239-248 are encompassed within the scope of this invention.Typically, such an insubstantial difference(s) involves substitution ofan amino acid with an amino acid having similar charge, hydrophobicity,or stereochemical characteristics. More drastic substitutions in FRregions, in contrast to CDR regions, may also be made as long as they donot adversely affect (e.g., reduce affinity by more than 50% as comparedto the unsubstituted binding protein) the binding properties of thebinding protein. Substitutions may also be made to germline the bindingprotein or stabilize its antigen-binding site.

Conservative modifications will produce molecules having functional andchemical characteristics similar to those of the molecule from whichsuch modifications are made. In contrast, substantial modifications inthe functional and/or chemical characteristics of the molecules may beaccomplished by selecting substitutions in the amino acid sequence thatdiffer significantly in their effect on maintaining (1) the structure ofthe molecular backbone in the area of the substitution, for example, asa sheet or helical conformation, (2) the charge or hydrophobicity of themolecule at the target site, and/or (3) the size of the molecule.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position (see, e.g., MacLennan et al. (1998)Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al. (1998) Adv.Biophys. 35:1-24).

Desired amino acid substitutions (whether conservative ornonconservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the moleculesequence, or to increase or decrease the affinity of the moleculesdescribed herein. Exemplary amino acid substitutions include, but arenot limited to, those set forth in Table 3.

TABLE 3 Exemplary Amino Acid Substitutions Original Exemplary MoreConservative Residues Substitutions Substitutions Ala (A) Val, Leu, IleVal Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C)Ser, Ala Ser Gln (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys,Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K) Arg, 1,4-diamino-butyricArg acid, Gln, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala,Tyr Leu Pro (P) Ala, Gly Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser SerTrp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met,Leu, Phe, Ala, Leu Norleucine

In certain embodiments, conservative amino acid substitutions alsoencompass normaturally occurring amino acid residues that are typicallyincorporated by chemical peptide synthesis rather than by synthesis inbiological systems.

In one embodiment, the method for making a variant V_(H) domaincomprises adding, deleting, or substituting at least one amino acid inthe disclosed V_(H) domains, or combining the disclosed V_(H) domainswith at least one V_(L) domain, and testing the variant V_(H) domain forIL-21R binding or modulation of IL-21R/IL-21 activity.

An analogous method for making a variant V_(L) domain comprises adding,deleting, or substituting at least one amino acid in the disclosed V_(L)domains, or combining the disclosed V_(L) domains with at least oneV_(H) domain, and testing the variant V_(L) domain for IL-21R binding ormodulation of IL-21R activity.

In some alternative embodiments, the anti-IL-21R binding proteins can belinked to a protein (e.g., albumin) by chemical cross-linking orrecombinant methods. The disclosed binding proteins may also be linkedto a variety of nonproteinaceous polymers (e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes) in manners set forth in U.S.Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and4,179,337. The binding proteins can be chemically modified by covalentconjugation to a polymer, for example, to increase their half-life inblood circulation. Exemplary polymers and attachment methods are shownin U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

The disclosed binding proteins can be modified to alter theirglycosylation; that is, at least one carbohydrate moiety can be deletedor added to the binding protein. Deletion or addition of glycosylationsites can be accomplished by changing amino acid sequence to delete orcreate glycosylation consensus sites, which are well known in the art.Another means of adding carbohydrate moieties is the chemical orenzymatic coupling of glycosides to amino acid residues of the bindingprotein, e.g., antibody (see, e.g., International ApplicationPublication No. WO 87/05330 and Aplin et al. (1981) CRC Crit. Rev.Biochem. 22:259-306). Removal of carbohydrate moieties can also beaccomplished chemically or enzymatically (see, e.g., Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52; Edge et al. (1981) Anal. Biochem.118:131; and Thotakura et al. (1987) Meth. Enzymol. 138:350).Modification of carbohydrate structures may be preferable as amino acidchanges in the Fc domain may enhance immunogenicity of a pharmaceuticalcomposition (see, e.g., International Application Publication No. WO2008/052030). For immunoglobulin molecules it has been demonstrated thatattachment of N-linked carbohydrate to Asn-297 of the CH2 domain iscritical for ADCC activity. Its removal enzymatically or throughmutation of the N-linked consensus site results in little to no ADCCactivity. In glycoproteins, carbohydrates may attach to the amidenitrogen atom in the side chain of an asparagine in a tripeptide motifAsn-X-Thr/Ser. This type of glycosylation, termed N-linkedglycosylation, commences in the endoplasmic reticulum (ER) with theaddition of multiple monosaccharides to a dolichol phosphate to form a14-residue branched carbohydrate complex. This carbohydrate complex isthen transferred to the protein by the oligosaccharyltransferase (OST)complex. Before the glycoprotein leaves the lumen of the ER, threeglucose molecules are removed from the 14-residue oligosaccharide. Theenzymes ER glucosidase I, ER glucosidase II and ER mannosidase areinvolved in ER processing. Subsequently, the polypeptides aretransported to the Golgi complex, where the N-linked sugar chains aremodified in many different ways. In the cis and medial compartments ofthe Golgi complex, the original 14-saccharide N-linked complex may betrimmed through removal of mannose (Man) residues and elongated throughaddition of N-acetylglucosamine (GlcNac) and/or fucose (Fuc) residues.The various forms of N-linked carbohydrates generally have in common apentasaccharide core consisting of three mannose and twoN-acetylglucosamine residues. Finally, in the trans Golgi, other GlcNacresidues can be added, followed by galactose (Gal) and a terminal sialicacid (Sial). Carbohydrate processing in the Golgi complex is called“terminal glycosylation” to distinguish it from “core glycosylation,”which takes place in the ER. The final complex carbohydrate units cantake on many forms and structures, some of which have two, three or fourbranches (termed biantennary, triantennary or tetraantennary). A numberof enzymes are involved in Golgi processing, including Golgimannosidases IA, IB and IC, GlcNAc-transferase I, Golgi mannosidase II,GlcNAc-transferase II, galactosyl transferase and sialyl transferase.

Methods for altering the constant region of a binding protein (such as,for example, the constant region of an antibody) are known in the art.Binding proteins with altered function (e.g., altered affinity for aneffector ligand such as FcR on a cell or the C1 component of complement)can be produced by replacing at least one amino acid residue in theconstant portion with a different residue (see, e.g., EuropeanApplication Publication No. EP 0 388 151 and U.S. Pat. Nos. 5,624,821and 5,648,260). Similar types of alterations could be described that, ifapplied to a murine or other species of binding protein, would reduce oreliminate similar functions.

For example, it is possible to alter the affinity of an Fc region of abinding protein (e.g., an IgG, such as a human IgG) for FcR (e.g., Fcgamma R1) or C1q. The affinity may be altered by replacing at least onespecified residue with at least one residue having an appropriatefunctionality on its side chain, or by introducing a charged functionalgroup, such as glutamate or aspartate, or perhaps an aromatic nonpolarresidue such as phenylalanine, tyrosine, tryptophan or alanine (see,e.g., U.S. Pat. No. 5,624,821).

For example, replacing residue 297 (asparagine) with alanine in the IgGconstant region significantly inhibits recruitment of effector cells,while only slightly reducing (about three-fold weaker) affinity for CIq(see, e.g., U.S. Pat. No. 5,624,821). The numbering of the residues inthe heavy chain is that of the EU index (see Kabat et al. (1991) supra).This alteration destroys the glycosylation site, and it is believed thatthe presence of carbohydrate is required for Fc receptor binding. Anyother substitution at this site that destroys the glycosylation site isbelieved to cause a similar decrease in lytic activity. Other amino acidsubstitutions, e.g., changing any one of residues 318 (Glu), 320 (Lys)and 322 (Lys), to Ala, are also known to abolish C1q binding to the Fcregion of IgG antibodies (see, e.g., U.S. Pat. No. 5,624,821).

Modified binding proteins can be produced which have a reducedinteraction with an Fc receptor. For example, it has been shown that inhuman IgG₃, which binds to the human Fc gamma R1 receptor, changing Leu235 to Glu destroys its interaction with the receptor. Mutations onadjacent or close sites in the hinge link region of a binding protein(e.g., replacing residues 234, 235 and 237 with Ala) can also be used toaffect binding protein affinity for the Fc gamma R1 receptor. Thenumbering of the residues in the heavy chain is based on the EU index(see Kabat et al. (1991) supra). Thus, in some embodiments of theinvention, the Fc region of the binding proteins of the inventioncontains at least one constant region mutation, such as, for example,changing Leu to Ala at position 234 (L234A), changing Leu to Ala atposition 235 (L235A), and/or changing Gly to Ala at position 237(G237A). In one embodiment, the Fc region of the binding proteincontains two constant region mutations, L234A and G237A (i.e.,“double-mutant” or “DM”). In another embodiment, the Fc region of thebinding protein contains three constant region mutations, L234A, L235A,and G237A (i.e., “triple-mutant” or “TM”). For example, a human IgGconstant region triple-mutant is set forth in SEQ ID NO:196.

Additional methods for altering the lytic activity of a binding protein,for example, by altering at least one amino acid in the N-terminalregion of the CH2 domain, are described in International ApplicationPublication No. WO 94/029351 and U.S. Pat. No. 5,624,821.

The binding proteins of the invention can be tagged with a detectable orfunctional label. These labels include radiolabels (e.g., ¹³¹I and⁹⁹Tc), enzymatic labels (e.g., horseradish peroxidase and alkalinephosphatase), and other chemical moieties (e.g., biotin).

The invention may also feature an isolated binding protein orantigen-binding fragment thereof that binds to IL-21R, in particular,human IL-21R. In certain embodiments, the anti-IL-21R binding proteinmay have at least one of the following characteristics: (1) it is amonoclonal or single specificity binding protein; (2) it is a humanbinding protein; (3) it is an in vitro generated binding protein; (4) itis an in vivo generated binding protein (e.g., transgenic mouse system);(5) it inhibits the binding of IL-21 to IL-21R; (6) it is an IgG1; (7)it binds to human IL-21R with an association constant of at least about10⁵ M⁻¹s⁻¹ (8) it binds to murine IL-21R with an association constant ofat least about 5×10⁴ M⁻¹s⁻¹; (9) it binds to human IL-21R with adissociation constant of about 10⁻³ (1/s) or less; (10) it binds tomurine IL-21R with a dissociation constant of about 10⁻² (1/s) or less;(11) it inhibits human IL-21R-mediated proliferation of humanIL-21R-expressing BaF3 cells with an IC₅₀ of about 1.75 nM or less; (12)it inhibits murine IL-21R-mediated proliferation of murineIL-21R-expressing BaF3 cells with an IC₅₀ of about 0.5 nM or less; (13)it inhibits human IL-21R-mediated proliferation of humanIL-21R-expressing TF1 cells with an IC₅₀ of about 14.0 nM or less; (14)it inhibits IL-21-mediated proliferation of human primary B cells withan IC₅₀ of about 1.9 nM or less; (15) it inhibits IL-21-mediatedproliferation of human primary CD4⁺ T cells with an IC₅₀ of about 1.5 nMor less; (16) it inhibits IL-21-mediated proliferation of murine primaryCD4⁺ T cells with an IC₅₀ of about 5.0 nM or less; (17) it has a meantotal body clearance of about 0.1-7.5 ml/hr/kg following, e.g., i.v.administration to animals, e.g., mammals, e.g., humans, nonhumanprimates, rodents; (18) it has a mean elimination half-life of about20-700 hr following, e.g., i.v., s.c., or i.p. administration toanimals, e.g., mammals, e.g., humans, nonhuman primates, rodents; (19)it has a mean steady-state volume of distribution of about 40-1500 ml/kgin animals, e.g., mammals, e.g., humans, nonhuman primates, rodents;(20) it has a bioavailability of about 35-100% following, e.g., s.c.administration to animals, e.g., mammals, e.g., humans, nonhumanprimates, rodents; (21) it has a mean dose-normalized AUC of about200-10,000 μg*hr/mL (per 1 mg/kg dosage) following, e.g., i.v., s.c., ori.p. administration to animals, e.g., mammals, e.g., humans, nonhumanprimates, rodents; (22) it has a mean dose-normalized C_(max)(maximumserum concentration) of about 0.5-30 μg/ml following, e.g., i.v., s.c.,or i.p. administration to animals, e.g., mammals, e.g., humans, nonhumanprimates, rodents; and (23) it modulates expression of IL-21 responsivecytokines or IL-21 responsive genes.

One of skill in the art will appreciate that the modifications describedabove are not exhaustive, and that many other modifications will beobvious to a skilled artisan in light of the teachings of the presentdisclosure.

Nucleic Acids, Cloning and Expression Systems

The disclosure provides isolated nucleic acids encoding the disclosedbinding proteins. The nucleic acids may comprise DNA or RNA, and theymay be synthetic (completely or partially) or recombinant (completely orpartially). Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesan RNA molecule with the specified sequence in which U is substitutedfor T.

Also contemplated are nucleic acids that comprise a coding sequence forone, two, or three CDRs, a V_(H) domain, a V_(L) domain, or combinationsthereof, as disclosed herein, or a sequence substantially identicalthereto (e.g., a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or higher identical thereto, or which is capable ofhybridizing under stringent conditions to the sequences).

In one embodiment, the isolated nucleic acids have nucleotide sequencesencoding heavy chain and light chain variable regions of an anti-IL-21Rbinding protein comprising at least one CDR chosen from the amino acidsequences of SEQ ID NOs:163-195, or a sequence encoding a CDR whichdiffers by one or two or three or four amino acids from the sequencesdescribed herein.

The nucleic acid can encode only the light chain or the heavy chainvariable region, or can encode a binding protein light or heavy chainconstant region, operatively linked to the corresponding variableregion. In one embodiment, the light chain variable region is linked toa constant region chosen from a kappa or a lambda constant region. Thelight chain constant region may also be a human kappa or lambda type. Inanother embodiment, the heavy chain variable region is linked to a heavychain constant region of a binding protein isotype chosen from IgG(e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA1, IgA2, IgD, and IgE. Theheavy chain constant region may be an IgG (e.g., an IgG1) isotype.

The nucleic acid compositions of the present invention, while often inthe native sequence (of cDNA or genomic DNA or mixtures thereof), exceptfor modified restriction sites and the like, can be mutated inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequences asdesired. In particular, nucleotide sequences substantially identical toor derived from native V, D, J, constant, switches and other suchsequences described herein are contemplated (where “derived” indicatesthat a sequence is identical to or modified from another sequence).

In one embodiment, the nucleic acid differs (e.g., differs bysubstitution, insertion, or deletion) from that of the sequencesprovided (e.g., by at least one but less than 10, 20, 30, or 40nucleotides; at least one but less than 1%, 5%, 10% or 20% of thenucleotides in the subject nucleic acid). If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences. The difference may be at a nucleotide(s) encoding anonessential residue(s), or the difference may be a conservativesubstitution(s).

The disclosure also provides nucleic acid constructs in the form ofplasmids, vectors, and transcription or expression cassettes, whichcomprise at least one nucleic acid as described herein.

The disclosure further provides a host cell that comprises at least onenucleic acid construct described herein.

Also provided is a method of making an encoded protein(s) from a nucleicacid(s) comprising the sequence(s) described herein. The methodcomprises culturing host cells under appropriate conditions so theyexpress the protein from the nucleic acid. Following expression andproduction, the V_(H) or V_(L) domain, or specific binding member, maybe isolated and/or purified using any suitable technique, and then usedas appropriate. The method can also include the steps of fusing anucleic acid encoding an scFv with nucleic acids encoding an Fc portionof a binding protein, and expressing the fused nucleic acid in a cell.The method can also include a step of germlining.

Antigen-binding fragments, V_(H) and/or V_(L) domains, and encodingnucleic acid molecules and vectors may be isolated and/or purified fromtheir natural environment, in substantially pure or homogenous form, or,in the case of nucleic acids, free or substantially free of nucleicacids or genes of origin other than the sequence encoding a polypeptidewith the require function.

Systems for cloning and expressing polypeptides in a variety of hostcells are known in the art. Cells suitable for producing bindingproteins are described in, for example, Fernandez et al. (1999) GeneExpression Systems, Academic Press. In brief, suitable host cellsinclude mammalian cells, insect cells, plant cells, yeast cells, orprokaryotic cells, e.g., E. coli. Mammalian cells available in the artfor heterologous polypeptide expression include lymphocytic cell lines(e.g., NSO), HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells,HeLa cells, baby hamster kidney cells, oocyte cells, and cells from atransgenic animal, e.g., mammary epithelial cells. In other embodiments,the nucleic acids encoding the binding proteins of the invention areplaced under the control of a tissue-specific promoter (e.g., amammary-specific promoter) and the binding proteins are produced intransgenic animals. For example, the binding proteins are secreted intothe milk of the transgenic animal, such as a transgenic cow, pig, horse,sheep, goat, or rodent.

Suitable vectors may be chosen or constructed to contain appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genes,and other sequences. The vectors may also contain a plasmid or viralbackbone. For details, see, e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2nd ed. 1989), Cold Spring Harbor Laboratory Press.Many established techniques used with vectors, including themanipulation, preparation, mutagenesis, sequencing, and transfection ofDNA, are described, e.g., in Current Protocols in Molecular Biology (2nded. 1992) Ausubel et al. eds., John Wiley & Sons.

A further aspect of the disclosure provides a method of introducing thenucleic acid into a host cell. For eukaryotic cells, suitabletransfection techniques may include calcium phosphate, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usinga retrovirus or other virus(es), e.g., vaccinia or baculovirus. Forbacterial cells, suitable techniques may include calcium chloridetransformation, electroporation, and transfection using bacteriophage.DNA introduction may be followed by a selection method (e.g., drugresistance) to select cells that contain the nucleic acid.

Uses of Anti-IL-21R Binding Proteins

Anti-IL-21R binding proteins that act as antagonists to IL-21R (e.g.,binding proteins, or antigen-binding fragments thereof, of the presentinvention) can be used to regulate at least one IL-21R-mediated immuneresponse, such as one or more of cell proliferation, cytokine expressionor secretion, chemokine secretion, and cytolytic activity, of T cells, Bcells, NK cells, macrophages, or synovial cells. Accordingly, thebinding proteins of the invention can be used to inhibit the activity(e.g., proliferation, differentiation, and/or survival) of an immune orhematopoietic cell (e.g., a cell of myeloid, lymphoid, or erythroidlineage, or precursor cells thereof), and, thus, can be used to treat avariety of immune disorders and hyperproliferative disorders of theblood. Examples of IL-21R-associated disorders/immune disorders that canbe treated include, but are not limited to, transplant rejection,graft-versus-host disease (GVHD), allergies (for example, atopicallergy), and autoimmune diseases. Autoimmune diseases include, but arenot limited to, diabetes mellitus, arthritic disorders (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis, and ankylosing spondylitis), spondyloarthropathy,multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosus (SLE), cutaneous lupus erythematosus, autoimmunethyroiditis, dermatitis (including atopic dermatitis and eczematousdermatitis), psoriasis, Sjögren's syndrome, IBD (including Crohn'sdisease and ulcerative colitis), asthma (including intrinsic asthma andallergic asthma), scleroderma and vasculitis. The binding proteins, orantigen-binding fragments thereof, of the present invention can be usedin methods to treat or prevent IL-21R-associated disorders/immunedisorders in subjects, e.g., in humans.

Multiple sclerosis is a central nervous system disease that ischaracterized by inflammation and loss of myelin sheaths (the fattymaterial that insulates nerves and is needed for proper nerve function).Inflammation that results from an immune response that is dependent onIL-21/IL-21R can be treated with the binding proteins and compositionsof this invention. In the experimental autoimmune encephalitis (EAE)mouse model for multiple sclerosis (see, e.g., Tuohy et al. (1988) J.Immunol. 141:1126-30; Sobel et al. (1984) J. Immunol. 132:2393-401; andTraugott (1989) Cell Immunol. 119:114-29), treatment of mice withinjections of an IL-21R antibody prior to (and continuously after)induction of EAE profoundly delayed the onset of the disease. Thebinding proteins, or antigen-binding fragments thereof, of the presentinvention can be used in methods to treat or prevent multiple sclerosisin subjects, e.g., in humans.

Arthritis is a disease characterized by inflammation in the joints.Rheumatoid arthritis (RA) is the most frequent form of arthritis,involving inflammation of connective tissue and the synovial membrane, amembrane that lines the joint. The inflamed synovial membrane ofteninfiltrates the joint and damages joint cartilage and bone. Studies showthat treatment of synovial cells and macrophages with IL-21 inducesthese cells to secrete cytokines and chemokines associated withinflammation. In the collagen-induced arthritis (CIA) mouse model forrheumatoid arthritis (see, e.g., Courtenay et al. (1980) Nature283:666-28; and Williams et al. (1995) Immunol. 84:433-39), treatment ofmice with IL-21 subsequent to CIA induction (and continuously)exacerbates the disease. Increased secretion of inflammatory cytokinesand chemokines, and more importantly, increased levels of diseaseresulting from immune responses that are dependent on IL-21, may betreated with the binding proteins of the invention. Similarly, thebinding proteins, or antigen-binding fragments thereof, of the presentinvention can be used in methods to treat or prevent RA or otherarthritic diseases in subjects, e.g., in humans.

Transplant rejection is the immunological phenomenon where tissues froma donor are specifically “attacked” by immune cells of the host. Theprinciple “attacking” cells are T cells, whose T cell receptorsrecognize the donor's MHC molecules as “foreign.” This recognitionactivates the T cell, which proliferates and secretes a variety ofcytokines and cytolytic proteins that ultimately destroy the transplant.T cells in a mixed lymphocyte reaction (MLR), an in vitro assay oftransplant rejection, proliferate more strongly when supplemented withIL-21. MLR and transplantation models have been described in CurrentProtocols in Immunology (2nd ed. 1994) Coligan et al. eds., John Wiley &Sons (see also Kasaian et al. (2002) supra; Fulmer et al. (1963) Am. J.Anat. 113:273-85; and Lenschow et al. (1992) Science 257:789-92). Thebinding proteins, or antigen-binding fragments thereof, of the presentinvention can be used in methods to reduce or prevent the MLR and/or totreat or prevent transplant rejection and related diseases (e.g., GVHD)that are dependent on IL-21 in subjects, e.g., in humans.

Systemic lupus erythematosus (SLE) is an autoimmune diseasecharacterized by the presence of autoantibodies, including antibodies toDNA, nuclear antigens, and ribonucleoproteins. These autoantibodies areassociated with tissue and organ damage. The cause of SLE is unknown,but the occurrence of autoantibodies suggests inadequate inhibition ofautoreactive T cells or B cells. The binding proteins, orantigen-binding fragments thereof, of the present invention can be usedin methods to inhibit the IL-21-mediated activities of autoreactive Tcells and B cells and/or to treat or prevent SLE or related diseases insubjects, e.g., in humans or in MRL-Fas^(lpr) mice (a mouse model forSLE) (Immunologic Defects in Laboratory Animals (1981) Gershwin et al.eds., Plenum Press).

The binding proteins, or antigen-binding fragments thereof, of thepresent invention also can be used in methods to treat or preventhyperproliferative disorders of the blood that are associated withaberrant activity of IL-21-responsive cells and IL-21R-responsive cellsin subjects, e.g., in humans. Examples of such cells include neoplasticcells of hematopoietic origin, e.g., cells arising from myeloid,lymphoid or erythroid lineages, or precursor cells thereof. Examples ofsuch neoplastic disorders include leukemic cancers and tumors of theblood, bone marrow (e.g., myeloma), and lymph tissue (e.g., lymphomas).In certain embodiments, the present invention is directed to thetreatment of various leukemic cancers including, but not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML) (reviewed in Vaickus (1991) Crit.Rev. Oncol./Hemotol. 11:267-97). Examples of lymphoid malignancies thatmay be treated by the present methods include, but are not limited to,acute lymphoblastic leukemia (ALL, which includes B-lineage ALL andT-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocyticleukemia (PLL), hairy cell leukemia (HCL), and Waldenstrom'smacroglobulinemia (WM). Additional forms of malignant lymphomas that canbe treated by the present invention include, but are not limited to,non-Hodgkin's lymphoma, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGL), Hodgkin's lymphoma, and variantsthereof.

In another aspect, the invention features a method of decreasing,inhibiting or reducing an acute phase response in a subject. An acutephase response is a response to inflammation, including the modulationof levels of acute phase proteins (e.g., C-reactive protein and serumalbumin). The method includes administering to the subject ananti-IL-21R binding protein or antigen-binding fragment thereof asdescribed herein, in an amount sufficient to decrease, inhibit or reducethe acute phase response in the subject. In one embodiment, the subjectis a mammal, e.g., a human suffering from an IL-21R-associated disorderas described herein, including, e.g., respiratory disorders,inflammatory disorders and autoimmune disorders.

Combination Therapy

In one embodiment, a pharmaceutical composition comprising at least oneanti-IL-21R binding protein or antigen-binding fragment thereof and atleast one therapeutic agent is administered in combination therapy. Thetherapy is useful for treating pathological conditions or disorders,such as immune and inflammatory disorders. The term “in combination” inthis context means that the binding protein composition and thetherapeutic agent are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second compound, the first of the two compoundsmay still be detectable at effective concentrations at the site oftreatment.

For example, the combination therapy can include at least oneanti-IL-21R binding protein, such as, for example, an anti-IL-21Rantibody, coformulated with, and/or coadministered with, at least oneadditional therapeutic agent. The additional agents may include at leastone cytokine inhibitor, growth factor inhibitor, immunosuppressant,anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor,cytotoxic agent, and/or cytostatic agent, as described in more detailbelow. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.Moreover, the therapeutic agents disclosed herein act on pathways thatdiffer from the IL-21/IL-21R pathway, and thus are expected to enhanceand/or synergize with the effects of the anti-IL-21R binding proteins.

Therapeutic agents used in combination with anti-IL-21R binding proteinsmay be those agents that interfere at different stages in the autoimmuneand subsequent inflammatory response. In one embodiment, at least oneanti-IL-21R binding protein described herein may be coformulated with,and/or coadministered with, at least one cytokine and/or growth factorantagonist. The antagonists may include soluble receptors, peptideinhibitors, small molecules, ligand fusions, antibodies (that bindcytokines or growth factors, or their receptors or other cell surfacemolecules), and “anti-inflammatory cytokines” and agonists thereof.

Examples of the agents that can be used in combination with theanti-IL-21R binding proteins described herein, include, but are notlimited to, antagonists of at least one interleukin (e.g., IL-1, IL-2,IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, and IL-22),cytokine (e.g., TNF-α, LT, EMAP-II, and GM-CSF), or growth factor (e.g.,FGF and PDGF). The agents may also include, but are not limited to,antagonists of at least one receptor for an interleukin, cytokine, orgrowth factor. Anti-IL-21R binding proteins can also be combined withinhibitors (e.g., antibodies) to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86(B7.2), CD90, or their ligands (e.g., CD154 (gp39, CD40L)), orLFA-1/ICAM-1 or VLA-4/VCAM-1 (see Yusuf-Makagiansar et al. (2002) Med.Res. Rev. 22(2):146-67)). Antagonists that can be used in combinationwith anti-IL-21R binding proteins described herein may includeantagonists of IL-1, IL-12, TNF-α, IL-15, IL-17, IL-18, IL-22, and theirreceptors.

Examples of IL-12 antagonists include antibodies that bind IL-12 (see,e.g., International Application Publication No. WO 00/056772); IL-12receptor inhibitors (e.g., antibodies to the IL-12 receptor), andsoluble IL-12 receptor and fragments thereof. Examples of IL-15antagonists include antibodies against IL-15 or its receptor, solublefragments of the IL-15 receptor, and IL-15-binding proteins. Examples ofIL-18 antagonists include antibodies to IL-18, soluble fragments of theIL-18 receptor, and IL-18 binding proteins (IL-18BP, Mallet et al.(2001) Circ. Res. 28). Examples of IL-1 antagonists includeInterleukin-1-Converting Enzyme (ICE) inhibitors (such as Vx740), IL-1antagonists (e.g., IL-1RA (anakinra, Amgen)), sIL-1RII (Immunex), andanti-IL-1 receptor antibodies.

Examples of TNF antagonists include antibodies to TNF (e.g., humanTNF-α), such as D2E7 (human anti-TNF-α antibody, U.S. Pat. No.6,258,562, HUMIRA™, BASF, Parsippany, N.J.), CDP-571/CDP-870/BAY-10-3356(humanized anti-TNF-α antibodies, Celltech/Pharmacia), cA2 (chimericanti-TNF-α antibody, REMICADE™, Centocor), and anti-TNF antibodyfragments (e.g., CPD870). Other examples include soluble TNF receptor(e.g., human p55 or p75) fragments and derivatives, such as p55kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein, LENERCEPT™) and 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL™, Immunex;see, e.g., Arthritis Rheumatism (1994) 37:S295 and J. Invest. Med.(1996) 44:235 A). Further examples include enzyme antagonists (e.g.,TNF-α converting enzyme inhibitors (TACE) such as alpha-sulfonylhydroxamic acid derivative (WO 01/055112) or N-hydroxyformamideinhibitor (GW 3333, -005, or -022)) and TNF-bp/s-TNFR (soluble TNFbinding protein; see, e.g., Arthritis Rheumatism (1996) 39(9):S284 andAm. J. Physiol. Heart Circ. Physiol. (1995) 268:37-42).

In other embodiments, the anti-IL-21R binding proteins described hereincan be administered in combination with at least one of the following:IL-13 antagonists, such as soluble IL-13 receptors and/or anti-IL-13antibodies; and IL-2 antagonists, such as IL-2 fusion proteins (e.g.,DAB 486-IL-2 and/or DAB 389-IL-2, Seragen (see, e.g., ArthritisRheumatism (1993) 36:1223)) and anti-IL-2R antibodies (e.g., anti-Tac(humanized antibody, Protein Design Labs (see Cancer Res. (1990)50(5):1495-502))). Another combination includes anti-IL-21R bindingproteins in combination with nondepleting anti-CD4 inhibitors such asIDEC-CE9.1/SB 210396 (anti-CD4 antibody, IDEC/SmithKline). Yet othercombinations include anti-IL-21R binding proteins with CD80 (B7.1) andCD86 (B7.2) costimulatory pathway antagonists (such as antibodies,soluble receptors, or antagonistic ligands), P-selectin glycoproteinligand (PSGL), and/or anti-inflammatory cytokines and agonists thereof(e.g., antibodies). The anti-inflammatory cytokines may include IL-4(DNAX/Schering, Palo Alto, Calif.), IL-10 (SCH 52000, recombinant IL-10,DNAX/Schering), IL-13, and TGF.

In other embodiments, at least one anti-IL-21R binding protein can becoformulated with, and/or coadministered with, at least oneanti-inflammatory drug, immunosuppressant, metabolic inhibitor, andenzymatic inhibitor. Nonlimiting examples of the drugs or inhibitorsthat can be used in combination with the IL-21R binding proteinsdescribed herein, include, but are not limited to, at least one of:nonsteroidal anti-inflammatory drugs (NSAID) (such as ibuprofen, tenidap(see, e.g., Arthritis Rheumatism (1996) 39(9):S280), naproxen (see,e.g., NeuroReport (1996) 7:1209-13), meloxicam, piroxicam, diclofenac,and indomethacin); sulfasalazine (see, e.g., Arthritis Rheumatism (1996)39(9):S281)); corticosteroids (such as prednisolone); cytokinesuppressive anti-inflammatory drugs (CSAID); and inhibitors ofnucleotide biosynthesis (such as an inhibitor of purine biosynthesis(e.g., a folate antagonist such as methotrexate) and an inhibitor ofpyrimidine biosynthesis (e.g., a dihydroorotate dehydrogenase (DHODH)inhibitor such as leflunomide (see, e.g., Arthritis Rheumatism (1996)39(9):S131 and Inflammation Research (1996) 45:103-7)).

Examples of additional inhibitors include at least one of:corticosteroids (oral, inhaled and local injection); immunosuppressants(such as cyclosporin and tacrolimus (FK-506)); mTOR inhibitors (such assirolimus (rapamycin) or a rapamycin derivative (e.g., an esterrapamycin derivative such as CCI-779 (see Elit (2002) Current OpinionInvestig. Drugs 3(8):1249-53 and Huang et al. (2002) Current OpinionInvestig. Drugs 3(2):295-304))); agents which interfere with thesignaling of proinflammatory cytokines such as TNF-α and IL-1 (e.g.,IRAK, NIK, IKK, p38, or a MAP kinase inhibitor); cox2 inhibitors (e.g.,celecoxib and variants thereof (MK-966); see, e.g., Arthritis Rheumatism(1996) 39(9):S81); phosphodiesterase inhibitors (such as R973401; see,e.g., Arthritis Rheumatism (1996) 39(9):S282); phospholipase inhibitors(e.g., an inhibitor of cytosolic phospholipase 2 (cPLA2) such astrifluoromethyl ketone analogs; see U.S. Pat. No. 6,350,892); inhibitorsof vascular endothelial cell growth factor (VEGF); inhibitors of theVEGF receptor; and inhibitors of angiogenesis.

The anti-IL-21R binding proteins disclosed herein can be used incombination with other therapeutic agents to treat specific immunedisorders as discussed in further detail below.

Nonlimiting examples of agents for treating arthritic disorders (e.g.,rheumatoid arthritis, inflammatory arthritis, rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis),with which an anti-IL-21R binding protein can be combined include atleast one of the following: TNF antagonists (such as anti-TNFantibodies); soluble fragments of TNF receptors (e.g., human p55 andp75) and derivatives thereof (such as p55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein, LENERCEPT™) and 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™)); TNF enzyme antagonists (such asTACE inhibitors); antagonists of IL-12, IL-15, IL-17, IL-18, and IL-22;T cell- and B cell-depleting agents (such as anti-CD4 or anti-CD22antibodies); small molecule inhibitors (such as methotrexate andleflunomide); sirolimus (rapamycin) and analogs thereof (e.g., CCI-779);cox2 and cPLA2 inhibitors; NSAIDs; p38, TPL-2, Mk-2, and NFκBinhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (suchas antibodies thereto and small molecule inhibitors); estrogen receptorbeta (ERB) agonists; and ERB-NFκB antagonists. Therapeutic agents thatcan be coadministered and/or coformulated with at least one IL-21/IL-21Rantagonist may include at least one of: a soluble fragment of a TNFreceptor (e.g., human p55 or p75) such as 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™); methotrexate; leflunomide; andsirolimus (rapamycin) or an analog thereof (e.g., CCI-779).

Nonlimiting examples of agents for treating multiple sclerosis withwhich an anti-IL-21R binding protein can be combined includeinterferon-β (for example, IFNβ-1a and IFNβ-1b), copaxone,corticosteroids, IL-1 inhibitors, TNF inhibitors, antibodies to CD40ligand, antibodies to CD80, and IL-12 antagonists.

Nonlimiting examples of agents for treating inflammatory bowel diseaseor Crohn's disease with which an anti-IL-21R binding protein can becombined include at least one of the following: budenoside; epidermalgrowth factor; corticosteroids; cyclosporine; sulfasalazine;aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;anti-IL-1 monoclonal antibodies; anti-IL-6 monoclonal antibodies; growthfactors; elastase inhibitors; pyridinyl-imidazole compounds; TNFantagonists as described herein; IL-4, IL-10, IL-13, and/or TGFβ oragonists thereof (e.g., agonistic antibodies); IL-11; glucuronide- ordextran-conjugated prodrugs of prednisolone, dexamethasone orbudesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides(ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1(TP10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate;antagonists of Platelet Activating Factor (PAF); ciprofloxacin; andlignocaine.

In other embodiments, an anti-IL-21R binding protein can be used incombination with at least one antibody directed at other targetsinvolved in regulating immune responses, e.g., transplant rejection orGVHD. Nonlimiting examples of agents for treating immune responses withwhich an IL-21/IL-21R antagonist can be combined include the followingantibodies against cell surface molecules (e.g., CD25 (IL-2 receptor α),CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1), CD86(B7-2), or combinations thereof). In another embodiment, an anti-IL-21Rbinding protein is used in combination with at least one generalimmunosuppressive agent, such as cyclosporin A or FK506.

Another aspect of the present invention relates to kits for carrying outthe combined administration of anti-IL-21R binding proteins with othertherapeutic agents. In one embodiment, the kit comprises at least oneanti-IL-21R binding protein formulated in a pharmaceutical carrier, andat least one therapeutic agent, formulated as appropriate in one or moreseparate pharmaceutical preparations.

Diagnostic Uses

The binding proteins of the invention may also be used to detect thepresence of IL-21R in biological samples. By correlating the presence orlevel of these proteins with a medical condition, one of skill in theart can diagnose the associated medical condition. For example,stimulated T cells increase their expression of IL-21R, and an unusuallyhigh concentration of IL-21R expressing T cells in joints may indicatejoint inflammation and possible arthritis. Illustrative medicalconditions that may be diagnosed by the binding proteins of thisinvention include, but are not limited to, multiple sclerosis,rheumatoid arthritis, and transplant rejection.

Binding protein-based detection methods, such as those commonly used forantibodies, are well known in the art, and include ELISA,radioimmunoassays, immunoblots, Western blots, flow cytometry,immunofluorescence, immunoprecipitation, and other related techniques.The binding proteins may be provided in a diagnostic kit thatincorporates at least one of these procedures to detect IL-21R. The kitmay contain other components, packaging, instructions, reagents, and/orother material to aid the detection of the protein and use of the kit.

Binding proteins may be modified with detectable markers, includingligand groups (e.g., biotin), fluorophores, chromophores, radioisotopes,electron-dense reagents, or enzymes. Enzymes are detected by theiractivity. For example, horseradish peroxidase is detected by its abilityto convert tetramethylbenzidine (TMB) to a blue pigment, quantifiablewith a spectrophotometer. Other suitable binding partners include biotinand avidin, IgG and protein A, and other receptor-ligand pairs known inthe art.

Binding proteins can also be functionally linked (e.g., by chemicalcoupling, genetic fusion, noncovalent association, or otherwise) to atleast one other molecular entity, such as another binding protein (e.g.,a bispecific or a multispecific binding protein), toxins, radioisotopes,cytotoxic or cytostatic agents, among others. Other permutations andpossibilities are apparent to those of ordinary skill in the art, andthey are considered equivalents within the scope of this invention.

Pharmaceutical Compositions and Methods of Administration

Certain embodiments of the invention include compositions comprising thedisclosed binding proteins. The compositions may be suitable forpharmaceutical use and administration to patients. The compositionscomprise a binding protein of the present invention and a pharmaceuticalexcipient. As used herein, “pharmaceutical excipient” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, etc., that are compatible withpharmaceutical administration. Use of these agents for pharmaceuticallyactive substances is well known in the art. The compositions may alsocontain other active compounds providing supplemental, additional, orenhanced therapeutic functions. The pharmaceutical compositions may alsobe included in a container, pack, or dispenser, together withinstructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. Pharmaceutical compositions may be topically or orallyadministered, or capable of transmission across mucous membranes.Examples of administration of a pharmaceutical composition include oralingestion or inhalation. Administration may also be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, cutaneous, ortransdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include at least one of the following components:a sterile diluent such as water, saline solution, fixed oils,polyethylene glycol, glycerine, propylene glycol, or other syntheticsolvent; antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate,citrate, or phosphate; and tonicity agents such as sodium chloride ordextrose. The pH can be adjusted with acids or bases by methods known inthe art. Such preparations may be enclosed in ampoules, disposablesyringes, or multiple dose vials.

Solutions or suspensions used for intravenous administration include acarrier such as physiological saline, bacteriostatic water, CREMOPHOREL® (BASF Corp., Ludwigshafen, Germany), ethanol, or polyol. In allcases, the composition must be sterile and fluid for easy syringability.Proper fluidity can often be obtained using lecithin or surfactants. Thecomposition must also be stable under the conditions of manufacture andstorage. Prevention of microorganisms can be achieved with antibacterialand antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbicacid, thimerosal, etc. In many cases, isotonic agents (sugar),polyalcohols (e.g., mannitol and sorbitol), or sodium chloride may beincluded in the composition. Prolonged absorption of the composition canbe accomplished by adding an agent that delays absorption, e.g.,aluminum monostearate or gelatin.

Oral compositions include an inert diluent or edible carrier. For thepurpose of oral administration, the binding proteins can be incorporatedwith excipients and placed, e.g., in tablets, troches, capsules, orgelatin. Pharmaceutically compatible binding agents or adjuvantmaterials can be included in the composition. The compositions maycontain (1) a binder such as microcrystalline cellulose, gum tragacanthor gelatin; (2) an excipient such as starch or lactose, (3) adisintegrating agent such as alginic acid, Primogel, or corn starch; (4)a lubricant such as magnesium stearate; (5) a glidant such as colloidalsilicon dioxide; and/or (6) a sweetening or flavoring agent.

The composition may also be administered by a transmucosal ortransdermal route. For example, binding proteins that comprise an Fcportion (for example, an antibody) may be capable of crossing mucousmembranes in the intestine, mouth, or lungs (via Fc receptors).Transmucosal administration can be accomplished by lozenges, nasalsprays, inhalers, or suppositories. Transdermal administration can beaccomplished with a composition containing ointments, salves, gels, orcreams known in the art. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used. Foradministration by inhalation, the binding proteins may be delivered inan aerosol spray from a pressured container or dispenser, which containsa propellant (e.g., liquid or gas), or a nebulizer.

In certain embodiments, the binding proteins of this invention areprepared with carriers to protect the binding proteins against rapidelimination from the body. Biodegradable polymers (e.g., ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid) are often used. Methods for the preparation of suchformulations are known by those skilled in the art. Liposomalsuspensions can be used as pharmaceutically acceptable carriers also.The liposomes can be prepared according to established methods known inthe art (see, e.g., U.S. Pat. No. 4,522,811).

The binding proteins or binding protein compositions of the inventionare administered in therapeutically effective amounts as described.Therapeutically effective amounts may vary with the subject's age,condition, sex, and severity of medical condition. Appropriate dosagescan be determined by a physician based upon clinical indications. Thebinding proteins or compositions may be given as a bolus dose tomaximize the circulating levels of binding proteins for the greatestlength of time. Continuous infusion may also be used.

As used herein, the term “subject” is intended to include human andnonhuman animals. Subjects may include a human patient having a disordercharacterized by cells that express IL-21R, e.g., a cancer cell or animmune cell. The term “nonhuman animals” of the invention includes allvertebrates, such as nonhuman primates, sheep, dogs, cows, chickens,amphibians, reptiles, etc.

Examples of dosage ranges that can be administered to a subject can bechosen from: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg/kg to 1 mg/kg,250 μg/kg to 2 mg/kg, 250 μg/kg to 1 mg/kg, 500 μg/kg to 2 mg/kg, 500μg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg, 10 mg/kg to 25 mg/kg,15 mg/kg to 25 mg/kg, 20 mg/kg to 25 mg/kg, and 20 mg/kg to 30 mg/kg (orhigher). These dosages may be administered daily, weekly, biweekly,monthly, or less frequently, for example, biannually, depending ondosage, method of administration, disorder or symptom(s) to be treated,and individual subject characteristics.

In certain circumstances, it may be advantageous to formulatecompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited for the patient. Each dosage unitcontains a predetermined quantity of binding protein calculated toproduce a therapeutic effect in association with the carrier. The dosageunit depends on the characteristics of the binding protein and theparticular therapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD_(5O)(the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED_(5O).Binding proteins that exhibit large therapeutic indices may be lesstoxic and/or more therapeutically effective.

The data obtained from the cell culture assays and animal studies can beused to formulate a dosage range in humans. The dosage of thesecompounds may lie within the range of circulating binding proteinconcentrations in the blood, which includes an ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage composition form employed and the route of administration. Forany binding protein used in the present invention, the therapeuticallyeffective dose can be estimated initially using cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofbinding protein that achieves a half-maximal inhibition of symptoms).The effects of any particular dosage can be monitored by a suitablebioassay. Examples of suitable bioassays include DNA replication assays,transcription-based assays, gene expression assays, IL-21/IL-21R bindingassays, and other immunological assays.

In one embodiment of the invention, a dose may be formulated in an exvivo whole blood cell assay. In such embodiment, a suitable bioassay fordetermining and monitoring a particular dosage includes a method ofdetermining a minimum serum concentration of an anti-IL-21R bindingprotein necessary to inhibit or reduce IL-21R activity, such asmodulation of expression of IL-21-responsive genes. In one embodiment ofthe invention, the IL-21-responsive gene is selected from the followingnonlimiting list: TNF, IFNγ, IL-6, IL-8, IL-10, CD19, STAT3, TBX21,CSF1, GZMB, PRF1, IL-2Rα, and IL-21R. In a preferred embodiment, theIL-21-responsive gene is selected from CD19, GZMB, IFNγ, IL-2Rα, IL6,and PRF-1. In a most preferred embodiment, the IL-21-responsive gene isIL-2Rα. Thus, the method of determining a minimum serum concentration ofan anti-IL-21R antibody necessary to inhibit or reduce IL-21R activitymay include determining a level of expression of more than oneIL-21-responsive gene, e.g., two, three, four, five, or sixIL-21-responsive genes.

The entire contents of all references, patent applications, and patentscited throughout this application are hereby incorporated by referenceherein.

EXAMPLES

The invention will be further illustrated in the following nonlimitingexamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art.

Example 1 Generation of Binding Proteins by Phage Display

The scFv parental clone 18A5, described in U.S. Pat. No. 7,495,085(incorporated by reference herein), was obtained from the CS human scFvlibrary by standard phage display methods, using BaF3 cells expressinghuman IL-21R as a target in rounds 1 and 3 and biotinylated IL-21R-Fcfusion protein as a target in round 2.

Example 2 Library Construction

Phage display libraries were based upon the parental 18A5 scFv, using apCANTAB6 vector in which the scFv was fused at its 3′ end to the intactgene III. Various CDR3 sequences were derived using techniques wellknown in the art.

Two overlapping blocks of six consecutive codons were randomized in theCDR3 of the V_(H) and the V_(L), producing a total of four libraries:H3B1, H3B2, L3B1, and L3B2. The following identify nucleotide and aminoacid sequences, respectively: IL-21R: 18A5 V_(H)CDR3 [SEQ ID NOs:199 and200]; H3B1 (library size 1.40×10⁹) [SEQ ID NOs:201 and 202]; H3B2(library size 1.00×10⁹) [SEQ ID NOs:203 and 204]; IL-21R: 18A5 V_(L)CDR3[SEQ ID NOs:205 and 206]; L3B1 (library size 9.00×10⁹) [SEQ ID NOs:207and 208]; L3B2 (library size 6.40×10⁹) [SEQ ID NOs:209 and 210].

Example 3 Phage Selection

All derivatives of 18A5 were isolated from the scFv libraries above byselection of phage able to bind in solution phase to biotinylated humanIL-21R extracellular domain His-Flag fusion proteins(“biotin-hIL-21R-H/F”) and biotinylated murine IL-21R extracellulardomain His-Flag fusion proteins (“biotin-mIL-21R-H/F”); all proceduresand techniques related to selection are well known to one of skill inthe art. A total of twenty-seven anti-IL-21R scFv were isolated by phageselection procedures.

Example 4 Library Screening

Resulting binding proteins in scFv format were chosen based on theirability to compete with parental 18A5 in human IgG1 format for bindingto biotin-hIL-21R-H/F and biotin-mIL-21R-H/F, to prevent thehIL-21-dependent proliferation of genetically engineered cell linesexpressing human IL-21R and the mIL-21-dependent proliferation ofgenetically engineered cell lines expressing murine IL-21R.

Example 4.1 Preparation of Crude Periplasmic Material (“Peri-Preps”) forUse in Screening Assays

Depending on the growth conditions used, scFv can be expressed insolution in the bacterial periplasmic space. To induce release of scFvinto the periplasm, 96-deep-well plates containing 990 μl 2×TY mediawith 0.1% glucose/100 μg/ml ampicillin were inoculated from thawedglycerol stocks (one clone per well) using the QPix2 Colony picker(Genetix, New Milton, England) and grown at 37° C. (999 rpm) for about 4hr. Cultures were induced with IPTG at a final concentration of 0.02 mMand grown overnight at 30° C. (999 rpm). The contents of the bacterialperiplasm (peri-preps) were released by osmotic shock. Briefly, plateswere centrifuged and pellets were resuspended in 150 μl TES periplasmicbuffer (50 mM Tris/HCl (pH 8.0)/1 mM EDTA (pH 8.0)/20% Sucrose),followed by the addition of 150 μl 1:5 TES:water, and incubated on icefor 30 min. Plates were centrifuged and the scFv-containing supernatantwas harvested.

Example 4.2 Epitope Competition Assay for Library Screening

Those scFv able to compete with the parental 18A5 antibody for bindingto human or murine IL-21R were identified from selected phage by ahomogeneous time-resolved fluorescence (HTRF®) assay. Purified parental18A5 antibody was covalently modified with cryptate, a derivative ofeuropium, according to the instructions in an HTRF® Cryptate LabelingKit (Cisbio, Bedford, Mass.). Peri-preps of scFv were prepared asdescribed above and diluted to 0.25% in PBS/0.4 M potassiumfluoride/0.1% BSA (HTRF® buffer); then 10 μl of the mixture wastransferred to the wells of black 384-shallow-well plates (Nunc,Rochester, N.Y.). Five μl of cryptate-conjugated 18A5 antibody was thenadded to each well, followed by 5 μl of a mixture of a 1:800 dilution ofstreptavidin-XL665 conjugate (Cisbio) and either 4.8 nMbiotin-hIL-21R-H/F or 40 nM biotin-mIL-21R-H/F. The mixture wasincubated for 2 hr at RT, and time-resolved fluorescence measurementswere made (340 nm excitation, 615 nm and 665 nm emission). Competitionwith 18A5 antibody was indicated by a reduction in thebackground-corrected ratio of emission at 665 nm to emission at 615 nm.

A total of 8280 independently isolated scFv were screened in the HTRF®assay using human IL-21R-H/F, and 376 clones able to compete with theparental 18A5 antibody for binding to biotin-hIL-21R-H/F were chosen forfurther analysis.

Example 5 DNA Sequence Analysis of Library-derived scFv—PCRAmplification of scFv Regions for Sequencing Analysis

The sequences of 287 18A5-derived scFv variants with improved IL-21Rbinding over that of the parent 18A5 scFv molecule were determined, andthe frequencies of amino acids found at each position were determined.Among the V_(H) clones, only two (1.7%) were derived from a librarywhich mutated the last six amino acids of, e.g., SEQ ID NO:169 (at theC-terminus of V_(H) CDR3), while the remainder were derived from alibrary which mutated the first six amino acids of, e.g., SEQ ID NO:169.Among the V_(L) clones, only one clone (0.6%) was derived from a libraryin which the last six amino acids of, e.g., SEQ ID NO:170 (at theC-terminus of V_(L) CDR3) were mutated, while the majority were derivedfrom alterations in the first six amino acids of, e.g., SEQ ID NO:170(at the N-terminus of V_(L) CDR3).

PCR amplification of scFvs was carried out using VENT® DNA Polymerase(New England Biolabs, Ispwich, Mass.) in FIN buffer (EpicentreBiotechnologies, Madison, Wis.) according to the manufacturer'sinstructions. Five μl of a 1:10 dilution of a stationary phase bacterialculture was used as the template for a final reaction volume of 20 μl.The cycling conditions used were a 2-min hot start at 94° C., 30 cyclesof denaturation at 94° C. (1 min), primer annealing at 55° C. (2 min)and extension at 72° C. (1 min), followed by a final extension at 72° C.(5 min). PCR products were verified by agarose gel electrophoresis andcleaned up with Exol/SAP (shrimp alkaline phosphatase) prior tosequencing with the M13rev primer.

The SEQ ID NOs for the CDR3 sequences of twenty-seven scFv are listed inTable 4. These scFv were chosen for further analysis based on assaysdescribed in Example 6.

TABLE 4 CDR3 SEQ ID NOs of Improved 18A5-derived scFv scFv Heavy CDR3Light CDR3 H3 165 170 H4 166 170 H5 167 170 H6 168 170 L1 169 171 L2 169172 L3 169 173 L4 169 174 L5 169 175 L6 169 176 L8 169 177 L9 169 178L10 169 179 L11 169 180 L12 169 181 L13 169 182 L14 169 183 L15 169 184L16 169 185 L17 169 186 L18 169 187 L19 169 188 L20 169 189 L21 169 190L23 169 191 L24 169 192 L25 169 193

Example 6 Characterization of Library-derived scFv Example 6.1Preparation of Purified scFv for Quantitative Analysis

Individual scFv clones were purified on a small scale by Ni-NTApurification on PHYTIP® columns (PhyNexus, Inc., San Jose, Calif.).Single colonies were grown in 20 ml 2×TY medium with 0.1% glucose/100μg/ml ampicillin in 50-ml conical tubes to mid-logarithmic phase at 37°C. with shaking at 250 rpm. Expression of scFv was induced with IPTG ata final concentration of 0.02 mM, and cultures were grown overnight at30° C. Cells were harvested by centrifugation and resuspended in 1 mlTES periplasmic buffer, followed by the addition of 1 ml 1:5 TES:waterand incubation on ice for 30 min. Lysates were centrifuged at 3200 rpmfor 10 min at 4° C., and supernatants were brought to 2 mM MgCl₂. scFvwere captured on Ni-NTA PHYTIPs® (PhyNexus) by repeated passage of thesupernatant over the PHYTIPs® on a Perkin Elmer (Waltham, Mass.)MINITRAK™ IX liquid handling robot, followed by washing in IMAC washbuffer and elution with 200 mM imidazole, 50 mM Tris, 300 mM NaCl (pH8.0). The buffer was exchanged to PBS by three cycles of dilution 1:10into PBS, followed by concentration on a 10,000 molecular weight cutofffilter plate (Millipore MULTISCREEN® ULTRACEL™ 96-well ultrafiltrationplate, Millipore, Billerica, Mass.). Samples were quantitated using aMicro BCA™ kit (Thermo Fisher Scientific Inc., Rockford, Ill.) using themanufacturer's bovine serum albumin standard.

Example 6.2 Assays for IL-21-Dependent Proliferation of CellsOverexpressing Human or Murine IL-21R

Inhibition assays were performed with 18A5-derived binding proteins(scFv and IgG) to measure their blockade of IL-21-dependentproliferation of cell lines transfected with human or murine IL-21R.BaF3 cells, a murine pre-B cell line, and TF1 cells, a human erythroidcell line, were retrovirally transduced with IL-21R and greenfluorescent protein (GFP). Cells were routinely grown in RPMI 1640 with10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin,and 0.00036% β-mercaptoethanol. Human IL-21R-BaF3 cell cultures weresupplemented with 50 ng/ml of human IL-21; murine IL-21R-BaF3 cellcultures were supplemented with 10 U/ml of IL-3; TF1 cell cultures weresupplemented with 50 ng/ml of GM-CSF. Prior to assay, cells were washed3× in assay medium lacking supplemental growth factors, resuspended inassay medium, and incubated at 37° C./5% CO₂ for 6 hr. To prepare assayplates, 5000 cells were added to the central 60 wells of a 96-wellflat-bottomed white tissue culture plate (Thermo Scientific, Waltham,Mass.) in a volume of 55 μl/well. Test scFv or IgG samples were preparedby diluting the stock sample in assay medium and diluting seriallythree-fold. Twenty-five μl of the binding protein samples were added tothe cells and incubated for 30 min at 37° C./5% CO₂. Twenty μl of assaymedium containing 100-400 pg/ml of human or murine IL-21 was added toeach well, and the cells were incubated for an additional 48 hr.Proliferation was measured by bringing plates to RT, adding 15 μl/wellCELLTITER-GLO®, incubating for 10 min at RT, and measuring luminescencewith a Perkin Elmer ENVISION™ plate reader. After purification withPhyNexus IMAC tips, 108 scFv were tested for neutralization ofIL-21-dependent proliferation of all three cell lines. All showedneutralization of human IL-21R-BaF3 cells, with IC₅₀s lower than orequal to that of the parental 18A5 scFv. A subset showed strongneutralization of proliferation of murine IL-21R-BaF3 cells and humanIL-21R-TF1 cells. Data from the 27 most potent clones are shown in FIGS.1-3, and are summarized in Table 5.

FIGS. 1-3 show the neutralization of proliferation by scFv of humanIL-21R-BaF3 cells (FIGS. 1 a-c); human IL-21R-TF1 cells (FIGS. 2 a-c);and murine IL-21R-BaF3 cells (FIGS. 3 a-c). Cells were mixed with theindicated scFv and incubated with 100 pg/ml (FIG. 1-2) or 400 pg/ml(FIG. 3) of human IL-21.

Example 6.3 Quantitative Epitope Competition Assay

Purified scFv were analyzed quantitatively for their ability to competewith the parental 18A5 antibody for binding to murine IL-21R in anenzyme-linked immunosorbent assay (ELISA). Parental 18A5 antibody wascoated overnight at 4° C. on 96-well Nunc MAXISORP® plates at aconcentration of 0.75 μg/ml in PBS. Plates were washed 3× using PBS, andthen blocked for 3 hr at RT in PBS/1% BSA/0.05% Tween-20. scFv weremixed with 36 nM biotinylated mIL-21R-H/F and incubated for 10 min atRT. Blocked plates were washed 3× with PBS, and 50 μl/well ofscFv/IL-21R mixtures were transferred to the appropriate plates andincubated for 1 hr at RT. Plates were washed 5× with PBS prior to theaddition of a 1:6000 dilution of horseradish peroxidase-conjugatedstreptavidin (Southern Biotech, Birmingham, Ala.) secondary antibody todetect bound biotinylated mIL-21R-H/F. Plates were then incubated for 1hr at RT and washed 7× with PBS. Signal was developed using3,3′,5,5′-tetramethylbenzidine (TMB), the reaction stopped with H₂SO₄,and the absorbance read at 450 nm on an ENVISION™ plate reader (PerkinElmer). 108 scFv purified by PhyNexus IMAC tips were tested in thisassay, and most competed with the parental 18A5 antibody for binding tobiotinylated murine IL-21R-H/F with IC₅₀s lower than that of theparental 18A5 scFv. Epitope competition data for the 27 clones with thehighest potencies in cell-based neutralization assays are shown in FIGS.4 a-c and summarized in Table 5.

TABLE 5 Neutralization of Human and Murine IL-21R in Cell-based Assaysand Competition with 18A5 Antibody for Murine IL-21R Binding IC₅₀ (nM)in Human IC₅₀ (nM) in Human IC₅₀ (nM) in Murine IC₅₀ (nM) in MurineIL-21R-BaF3 IL-21R-TF1 IL-21R-BaF3 IL-21R Epitope Neutralization AssayNeutralization Assay Neutralization Assay Competition ELISA H3 7.7 98.125.68 14 H4 3.8 9.3 nd nd H5 7.9 178.5 53.66 17 H6 13.8 150 (estimated)nd 13 L1 3.7  55 (estimated) 28.77 7 L2 3.1 37.5 2.41 5 L3 27.6   7(estimated) 13.78 100 L4 2.1  60 (estimated) nd 8 L5 2.1  20 (estimated)38.52 7 L6 5.9 150 (estimated) 0.29 4 L8 4.1 51.3 715.27 7 L9 2.8 27.73.61 7 L10 15.1 7 nd 40 L11 4.2 38.3 10.03 6 L12 2.6 54.9 87.77 8 L1311.0 257.4 1.25 7 L14 3.2 33.5 6.49 6 L15 3.3 30.3 53.49 14 L16 3.7 67.44.71 6 L17 1.6 60.3 2.66 12 L18 3.7 54.4 8.34 8 L19 4.5 35.3 13.59 15L20 3.1 57.5 15.39 5 L21 9.4 100 (estimated) 162.27 28 L23 1.5 15.3 nd12 L24 2.4 18.7 3.73 6 L25 3.7 33.1 15.55 9

Example 7 Conversion of Parental 18A5 IgG to Germline Sequence

The following fifteen scFv with modified V_(L) regions, along with thegermlined parental 18A5 V_(L) (see below), were chosen for conversion tofull-length human IgG lambda: L2, L3, L6, L9, L11, L13, L14, L16, L17,L18, L19, L20, L23, L24, and L25. Four scFv with modified V_(H) regions,H3, H4, H5, and H6, along with the germlined parental 18A5 V_(H) (seebelow) were chosen for conversion to full-length human IgG1.

The V_(H) and V_(L) amino sequences of the parental 18A5 antibody weremodified so that the sequences outside the CDR regions matched theclosest human germline sequences: DP67/VH4B+(VBASE_AA:WAPOOCEAZ_(—)1)and JH1/JH4/JH5 in the case of the V_(H), and DPL16/VL3.1(VBASE_AA:WAPOOCEMI_(—)1) in the case of the V_(L). Modifications weredone by a combination of gene synthesis at GENEART (Regensburg, Germany)and site-directed changes introduced by PCR. In addition, the sequenceswere codon-optimized for expression in mammalian cells by GENEART usingtheir proprietary methods. An alignment of the parental 18A5 sequencesand the germline-corrected 18A5 sequences is shown below:

18A5 Heavy Chain Comparison

18A5 Light Chain Comparison

Germline-Corrected V_(H) Sequence (Changes from Parental Sequence areBold and Underlined):

(SEQ ID NO: 6) Parental QVQLQESGPGLVKTSETLSLTCAVSGYSISSGYYWGWIRQPPGKG(SEQ ID NO: 8) Germlined QVQLQESGPGLVK P SETLSLTCAVSGYSISSGYYWGWIRQPPGKGParental LEWIGSISHTGNTYYNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGGISRPGermlined LEWIGSISHTGNTYYNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGGISRPParental EYWGKGTLVTVSS Germlined EYWG Q GTLVTVSSGermline-Corrected V_(L) Sequence (Changes from Parental Sequence areBold and Underlined):

(SEQ ID NO: 10) Parental SSELTQDPPVSVALGQTVTLTCQGDSLRTYYASWYQQKSGQAPIL(SEQ ID NO: 12) Gemlined SSELTQDP A VSVALGQTV RI TCQGDSLRTYYASWYQQK PGQAP V L ParentalLLYGKHKRPSGIPDRFSGSTSGDTASLTITGAQAEDEADYYCNSRDSSGNPHVLFGGGTQ GermlinedVI YGKHKRPSGIPDRFSGS S SG N TASLTITGAQAEDEADYYCNSRDSSGNPHVLFGGGTQParental LTVL Germlines LTVL

Example 8 Conversion of Library-Derived scFv to IgG

The CDR3 regions of the V_(L) and V_(H) domains of improved 18A5 scFvderivatives were amplified by PCR and subcloned into thegermline-corrected V_(L) and V_(H) frameworks of the parental 18A5 bythe following method. A PCR fragment encompassing the 5′ portion of thegermlined 18A5 V_(H) gene was generated by amplification of the plasmidpSMED2_OP18A5G_huIgG1 with primers BssHII_II_V_(H) _(—) F(5′-GCTTGGCGCGCACTCTCAGGTGCAGCTGCAGGAG-3′) [SEQ ID NO:230] and GV_(H)_(—) R_for_BssHII (5′-TCAGGGAGAACTGGTTCTTGG-3′) [SEQ ID NO:231]. A PCRfragment encompassing the 3′ portion of the V_(H) gene from the improvedscFv clone VH3 was amplified with the following primers: G_V_(H) _(—)F_for_SalI (5′-TCCAAGAACCAGTTCTCCCTG-3′) [SEQ ID NO:232] andscFv_SalI_V_(H) _(—) R(5′-GCGACGTCGACAGGACTCACCACTCGAGACGGTGACCAGGGTGCC-3′) [SEQ ID NO:233].Fragments were gel-purified, and then the two were mixed and amplifiedwith the outside primer sets BssHII_G_V_(H) _(—) F and SalI_V_(H) _(—) Rto generate a complete V_(H) gene fragment. This was digested withBssHII and SalI and ligated into a vector containing the constantregions of human IgG1 with a triple-mutant hinge region. The insert wasreamplified with BssHII_II_V_(H) _(—) F and a new primer (Sal_V_(H) _(—)R_RJ (5′-GCGACGTCGACAGGACTCACCACTCGAGACGG-3′)) [SEQ ID NO:234] in orderto alter the coding sequence of the V_(H) J segment to conform to theJH1 germline sequence, and ligated into a human IgG1-triple-mutantconstant region vector.

The V_(L) genes from improved scFv were subcloned by a similar method. APCR fragment encompassing the 5′ portion of the 18A5 V_(L) gene wasgenerated by amplification of the plasmid pSMEN2_OP18A5G_hu Lambda withprimers BssHII_II_V_(L) _(—) F(5′-GCTTGGCGCGCACTCTTCCTCTGAGCTGACCCAG-3′) [SEQ ID NO:235] andscFv_V_(L) _(—) R_for_BssHII (5′-GCCTGAGCCCCAGTGATGGTCA-3′) [SEQ IDNO:236]. PCR fragments encompassing the 3′ portions of the V_(L) genesfrom improved scFv clones were amplified with the primers GV_(L) _(—)F_for_XbaI (5′-ACCGCCTCCCTGACCATCAC-3′) [SEQ ID NO:237] andscFv_XbaI_V_(L) _(—) R(5′-GCGCCGTCTAGAGTTATTCTACTCACCTAAAACGGTGAGCTGGGTCCC TC-3′) [SEQ IDNO:238]. Fragments were gel-purified, and then fragments correspondingto the 5′ and 3′ portions of each gene were mixed and amplified with theoutside primer set BssHII_II_V_(L) _(—) F and scFv_XbaI_V_(L) _(—) R togenerate complete V_(L) gene fragments. These were digested with BssHIIand XbaI, and ligated into a vector containing the constant regions ofthe human lambda gene.

Example 9 Characterization of Improved IgG In Vitro Example 9.1Transient Small-Scale Expression of Binding Proteins

Clones were tested for function in full IgG format following transientexpression in cos-7 cells. Each light chain in the set of sixteen testsequences (germlined parental 18A5 V_(L) and L2, L3, L6, L9, L11, L13,L14, L16, L17, L18, L19, L20, L23, L24 and L25) was paired with eachheavy chain in the set of five test sequences (H3, H4, H5, and H6, alongwith V_(H) _(—) P, the germlined parental 18A5 V_(H) domain). Eachplasmid in the pair (1.4 μg) was combined with the TRANSIT® transfectionreagent (Minis, Madison, Wis.) according to the manufacturer'sinstructions, and DNA:TRANSIT® reagent complexes were added tomonolayers of cos-7 cells growing in Dulbecco's Modified Eagle's medium(DMEM)/10% heat-inactivated fetal bovine serum/penicillin/streptomycin/2mM L-glutamine in 6-well tissue culture plates. After 24 hr, the mediumwas changed to a serum-free medium (R1CD1), and was then collected 48 hrlater. Binding proteins, now comprising full-length antibodies, werequantitated by anti-human IgG ELISA.

Example 9.2 Activity of Anti-IL-21R IgG in Neutralization of CellProliferation

The 80 transiently expressed IgGs in serum-free conditioned medium weretested for activity in IL-21-dependent proliferation assays in threecell lines as described above: (1) human IL-21R-BaF3 cells, (2) murineIL-21R-BaF3 cells, and (3) human IL-21R-TF1 cells. All 80 pairs showedneutralization of proliferation of human IL-21R-expressing BaF3 cells,and all pairs except those involving VH4 showed neutralization of humanIL-21R-expressing TF1 cells (data not shown). All 80 pairs also showedneutralization of proliferation of murine IL-21R-expressing BaF3 cells,with the strongest neutralization generally associated with light chainspaired with the parental heavy chain and the weakest neutralizationgenerally associated with the VH4 heavy chain (data not shown).Neutralization data from the most potent 21 IgG combinations (AbA-AbU)are shown in FIG. 5, and IC₅₀ data are summarized in Table 6.

Assays were conducted on human IL-21R-BaF3 cells with 100 pg/ml of humanIL-21 (FIGS. 5 a-c), human IL-21R-TF1 cells with 100 pg/ml of humanIL-21 (FIGS. 5 d-f), or murine IL-21R-BaF3 cells with 400 pg/ml ofmurine IL-21 (FIGS. 5 g-i). IL-21 was added to the cells after theindicated antibodies; proliferation was measured with CELLTITER-GLO®after 48 hr. FIGS. 26 a-c show additional studies demonstrating similarinhibition in the same three cell lines.

Example 9.3 Anti-IL-21R IgG Binding to Transiently Expressed Rat andCynomolgus Monkey IL-21R

A subset of binding proteins was tested for binding to rat, cynomolgusmonkey, human IL-21R, or human IL-2R-γ common subunit expressedtransiently on the surfaces of CHO-PA-Dukx cells. Cells were transfected48 hr prior to the assay. On the day of the assay, cells were washedgently 5× in PBS containing 0.9 mM CaCl₂ and 0.45 mM MgCl₂ (PBS/CaMg) onan automated plate washer (Titertek, Huntsville, Ala.), and blocked for1 hr at RT in PBS/CaMg/5% nonfat dry milk. Conditioned media fromtransiently expressed anti-IL-21R IgGs were serially diluted in blockingbuffer and added to the cells in the blocked plates for 1 hr at RT.Cells were washed 5× with PBS/CaMg and then incubated with horseradishperoxidase-conjugated anti-human IgG for 1 hr at RT. Cells were thenwashed 10× in PBS/CaMg and all of the wash buffer was removed. Cellswere incubated with 100 μl TMB until the color reaction reachedsaturation, stopped with 100 μl of 0.18 M H₂SO₄, and read at A450 on aPerkin Elmer ENVISION™ plate reader.

All of the twenty-one IgGs bound to CHO cells transiently expressinghuman (FIGS. 6 a-c), rat (FIGS. 6 d-f), or cynomolgus monkey (FIGS. 6g-i) IL-21R. Most showed no binding above background to a controlprotein (human gamma (γ) common chain) transiently expressed on CHOcells, but a subset of IgGs (AbD, AbE, AbF, AbH, AbL, and AbM) boundabove background at 13 nM or greater (FIGS. 6 j-l). Data are summarizedin Table 6.

TABLE 6 Summary of Neutralization of Human and Murine IL-21R Activity inCell-proliferation Assays and Binding to Human, Rat, and CynomolgusMonkey IL-21R Expressed on CHO Cells Human Rat Monkey Human IL-21RIL-21R IL-21R γ-Common Human Human Murine Binding Binding BindingBinding IL-21R- IL-21R- IL-21R- (13 nM Ab (13 nM Ab (13 nM Ab (13 nM AbBaF3 TF1 BaF3 in Cell in Cell in Cell in Cell Binding ProliferationProliferation Proliferation ELISA; ELISA; ELISA; ELISA; Protein IC₅₀(nM) IC₅₀ (nM) IC₅₀ (nM) A450) A450) A450) A450) AbA 0.97 3.80 0.081.196 1.124 1.352 0.111 AbB 1.14 3.34 0.421 1.147 1.09 1.333 0.107 AbC0.82 3.36 0.03 1.218 0.999 1.277 0.137 AbD 0.91 2.67 0.01 1.247 0.8741.375 0.197 AbE 0.56 2.28 0.04 1.257 1.111 1.423 0.223 AbF 0.54 2.410.304 1.347 1.001 1.458 0.433 AbG 0.77 3.84 0.07 1.35 1.112 1.304 0.108AbH 0.94 3.64 0.327 1.35 1.097 1.324 0.152 AbI 1.00 3.80 0.224 1.2371.088 1.209 0.107 AbJ 0.65 4.60 0.4 1.217 1.261 1.273 0.126 AbK 0.984.00 0.079 1.364 1.175 1.338 0.108 AbL 0.68 4.25 0.227 1.454 1.257 1.5140.219 AbM 1.08 4.22 0.125 1.197 0.78 1.45 0.224 AbN 0.50 1.59 0.4351.214 0.702 1.497 0.136 AbO 0.52 2.91 0.065 1.107 1.101 1.358 0.108 AbP0.75 3.48 0.03 1.308 1.03 1.313 0.112 AbQ 0.68 4.62 0.153 1.255 1.1611.31 0.125 AbR 0.87 3.94 0.302 1.334 1.108 1.35 0.109 AbS 1.53 5.00 0.041.017 1.166 1.224 0.118 AbT 0.67 3.26 0.093 1.078 0.994 1.219 0.102 AbU0.73 3.13 0.184 1.289 0.927 1.314 0.104

Example 9.4 BIACORE™ Analysis of Selectivity of Anti-IL-21R IgG Bindingto Human IL-21R

The specificity of binding of a subset of transiently expressedanti-IL-21R binding proteins (here antibodies) was tested on a BIACORE™2000 surface plasmon resonance instrument. Anti-human-IgG, anti-murineimmunoglobulin antibodies, and murine IL-21R-H/F were immobilized onto aresearch-grade carboxymethyl-dextran chip (CM5) using standard aminecoupling. The sensor chip surface was activated with EDC/NHS for 7 minat a flow rate of 20 μl/min. The first flow cell was used as referencesurface to correct for bulk refractive index, matrix effects, andnonspecific binding. Capture antibodies (7,150 resonance units (RU) ofanti-human-Fc antibody (Invitrogen Corporation, Carlsbad, Calif.) onflow cell 2 and 7,500 RU of anti-murine-Fc antibody on flow cell 3) werediluted to 10 μg/ml in sodium acetate buffer (pH 5.0) and injected overthe activated surface. Remaining activated groups were blocked with 1.0M ethanolamine (pH 8.0). The molecular weights of the anti-human IgG andthe anti-murine IgG were both 150 kD, and the molecular weight of theIL-21R monomer was 27 kD.

Conditioned media containing anti-IL-21R antibodies and antibodycontrols (murine anti-human IL-2Rβ and murine anti-human IL-4R(R&DSystems, Minneapolis, Minn.); human anti-human IL-13 (Wyeth, Cambridge,Mass.)) were diluted in HBS/EP buffer supplemented with 0.2% bovineserum and injected onto all four flow cells of the BIACORE™ chip,capturing 500-700 (RU) of antibody on the species-appropriate captureantibody. Following a 5 sec washing period, 50 nM solutions of apositive control protein (murine IL-21R-H/F), two human proteins relatedto IL-21R (human IL-2Rβ and human sIL-4R(R&D Systems)), or an unrelatedHis/FLAG-tagged protein (human IL-13-H/F) were injected over thecaptured antibodies on the chip. The association and dissociation phaseswere monitored for 120 and 180 sec, respectively, followed by two 5 μlinjections of glycine (pH 1.5) to regenerate a fully active capturingsurface. All binding experiments were done at 25° C. in HBS/EP buffer.Blank and buffer effects were subtracted for each sensorgram usingdouble referencing.

All of the anti-IL-21R antibodies tested (18A5 antibody and AbA-AbU)showed clear binding to murine IL-21R, but no binding to theIL-21R-related proteins human IL-2Rβ and human soluble IL-4R, or to theunrelated His/FLAG-tagged protein human IL-13-His/FLAG (FIGS. 7 a-c).Controls indicated that IL-2Rβ and human soluble IL-4R could be capturedby specific anti-IL-2Rβ and anti-IL-4R antibodies (FIG. 7 d).

Example 9.5 Purification of Transiently Expressed Antibodies

Seven antibodies (human IgG1 triple-mutant versions: AbS, AbT, AbO, AbP,and AbU; and double-mutant versions: AbQ and AbR) were transientlyexpressed in cos-7 cells and purified for further analysis. In addition,three versions of AbT with human IgG tails expected to have differentlevels of Fc receptor binding (wild-type IgG1, IgG4, and IgG1double-mutants) were also prepared. The TRANSIT® protocol describedabove was followed, except that 25 μg of each plasmid was used totransfect cells in each of eight T-175 flasks. Following the firstharvest of conditioned medium, fresh R1CD1 was added and then collectedafter an additional 72 hr. Conditioned media were pooled and filtered ona 0.22 μm filter. Antibodies were loaded onto protein A resin, elutedwith 20 mM citric acid/150 mM sodium chloride (pH 2.5), neutralized withTris (pH 8.5), and dialyzed into PBS.

Example 9.6 BIACORE™ Analysis of Antibody Binding to Human and MurineIL-21R

The kinetics of binding of anti-IL-21R antibodies to human and murineIL-21R-H/F was tested on a BIACORE™ surface plasmon resonanceinstrument. Anti-human IgG antibodies (Invitrogen Corporation) wereimmobilized onto a research-grade carboxy-methyl-dextran chip (CM5)using standard amine coupling. The surface was activated with EDC/NHSfor 7 min at a flow of 20 μl/min. The first flow cell was used as areference surface to correct for bulk refractive index, matrix effects,and nonspecific binding. The anti-human-Fc antibody was diluted to 20μg/ml in 10 mM sodium acetate buffer (pH 5.0), and 2950-3405 resonanceunits (RU) were captured on each of the four flow cells. Remainingactivated groups were blocked with 1.0 M ethanolamine-HCl (pH 8.5).

Anti-IL-21R antibodies were diluted to 0.1-0.2 μg/ml in HBS/EP buffersupplemented with 0.2% bovine serum albumin and loaded onto the BIACORE™chip. Following a brief washing period, solutions of 0-100 nM humanIL-21R-H/F or 10-500 nM murine IL-21R-H/F were injected over the chip ata flow rate of 50 μl/min. The association phase was run for 3 min forhuman and murine IL-21R kinetics, and the dissociation phase wasmonitored for 15 min for hIL-21R and for 5 min for mIL-21R, followed bytwo 10 μl injections and one 30 μl injection of glycine (pH 1.5), toregenerate a fully active capturing surface. All binding experimentswere done at 25° C. in HBS/EP buffer, and the sample rack was kept at15° C. Blank and buffer effects were subtracted for each sensorgramusing double referencing. Sensorgrams are shown in FIGS. 8 a-b (humanIL-21R-His/FLAG) and FIGS. 8 c-d (murine IL-21R-His/FLAG). Bindingkinetic parameters are shown in Table 7A, and additional kinetic datafrom a replicate experiment are shown in Table 7B.

In addition, AbS and AbT were tested for binding kinetics to cynomolgusmonkey IL-21R-His/FLAG by the above-described protocol. Binding profilesto human and cynomolgus monkey IL-21R-H/F were similar for both AbS andAbT (FIG. 9). FIG. 9 shows cynomolgus monkey IL-21R-His/FLAG binding toAbS (9 a); and to AbT (9 c); and human IL-21R-His/FLAG binding to AbS (9b); and AbT (9 d).

TABLE 7A Kinetic Parameters of Anti-IL-21R Antibody Binding Human andMurine IL-21R-His/FLAG Human IL-21R Murine IL-21R ka kd KD ka kd KDAntibody (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 18A5 2.43E+05 1.08E−034.43E−09 2.12E+05 1.53E−02 7.20E−08 AbO 2.41E+05 1.14E−04 4.75E−101.12E+05 5.49E−03 4.92E−08 AbP 1.94E+05 1.19E−04 6.15E−10 9.99E+045.08E−03 5.08E−08 AbQ 4.39E+05 9.34E−05 2.13E−10 3.01E+05 2.07E−026.88E−08 AbR 1.70E+05 9.61E−05 5.67E−10 7.65E+04 4.93E−03 6.45E−08 AbS1.44E+05 2.91E−04 2.02E−09 1.99E+05 3.32E−03 1.67E−08 AbT 1.79E+056.78E−05 3.79E−10 2.11E+05 3.31E−03 1.57E−08 AbU 1.86E+05 8.18E−054.40E−10 9.81E+04 4.34E−03 4.42E−08

TABLE 7B Kinetic Parameters of Anti-IL-21R Antibody Binding HumanIL-21R-His/FLAG Human IL-21R ka kd KD Antibody (1/Ms) (1/s) (M) 18A53.04E+05 1.34E−03 4.40E−09 AbP 2.33E+05 1.02E−04 4.36E−10 AbQ 4.39E+059.34E−05 2.13E−10 AbR 2.48E+05 9.76E−05 3.94E−10 AbS 2.02E+05 3.05E−041.51E−09 AbT 2.73E+05 7.42E−05 2.72E−10 AbU 2.38E+05 7.83E−05 3.29E−10

Example 9.7 BIACORE™ Epitope Competition Assay

Antibodies AbS and AbT and the parental antibody 18A5 were immobilizeddirectly onto a CM5 BIACORE™ chip. Murine IL-21R-H/F (100 nM) wasallowed to flow over the chip for 300 sec, followed by a wash (100 sec),and then a 5 μg/ml solution of either AbS, AbT, D5, or a normeutralizinganti-mIL-21R antibody (7C2) was allowed to flow over the surface. Noadditional binding was observed with AbS, AbT, and D5, indicating thattheir binding site on mIL-21R-H/F was blocked by concurrent binding toAbS, AbT, or 18A5 antibody (FIG. 10 a). In contrast, the normeutralizingcontrol anti-IL-21R antibody 7C2 was able to bind to mIL-21R-H/Fcaptured on AbS, AbT, or 18A5 antibody, indicating that this controlantibody bound at a different epitope from the one bound by the captureantibodies.

Similarly, AbS and AbT did not bind to human IL-21R-H/F captured by AbSor AbT immobilized on a CM5 BIACORE™ chip, while the control anti-humanIL-21R antibody (9D2) was able to bind human IL-21R-H/F captured by AbSor AbT (FIG. 10 b). This observation suggested that the binding site forAbS is blocked by concurrent binding by AbT, and vice versa.

Example 9.8 Cell-Based Proliferation Assays

Purified IgGs were tested for activity in IL-21-dependent proliferationassays in three cell lines as described above: human IL-21R-BaF3 cells,murine IL-21R-BaF3 cells, and human IL-21R-TF-1 cells. All showed stronginhibition of both human and murine IL-21R-dependent proliferation withgreater potency than that of the parental 18A5 IgG (FIG. 11, Table 8).Assays were conducted on human IL-21R-BaF3 cells with 100 pg/ml of humanIL-21 (FIG. 11 a), murine IL-21R-BaF3 cells with 200 pg/ml of murineIL-21 (FIG. 11 b), and human IL-21R-TF-1 cells with 100 pg/ml of humanIL-21 (FIG. 11 c). FIG. 26 d depicts the results of an additional studyof the effects of these antibodies on human IL-21R-BaF3 cells.

TABLE 8 Neutralization of Proliferation of Human IL-21R-BaF3 Cells,Murine IL-21R-BaF3 Cells, and Human IL-21R-TF-1 Cells Human Murine HumanIL-21R-BaF3 IL-21R-BaF3 IL-21R-TF1 Neutralization IC₅₀ NeutralizationIC₅₀ Neutralization IC₅₀ Antibody (nM) (nM) (nM) 18A5 antibody 1.71177.23 13.99 AbR 0.56 0.34 1.63 AbS 0.68 0.04 6.67 AbT 0.30 0.05 2.32AbX 0.54 nd nd IL21R-Fc 0.20 (human 0.04 (mouse 7.22 (human IL-21R-Fc)IL-21R-Fc) IL-21R-Fc)

Example 9.9 Primary Human B cell Proliferation Assays

Anti-IL-21R antibodies were tested for their ability to inhibitIL-21-dependent proliferation of primary human B cells. Buffy coat cellsfrom healthy human donors were obtained from Massachusetts GeneralHospital (Boston, Mass.). The cells were incubated with a ROSETTESEP™ Bcell enrichment cocktail (StemCell Technologies, Vancouver, Canada), andB cells isolated according to the manufacturer's instructions. Theresulting population (60-80% CD19⁺ B cells) were cultured in RPMIcontaining 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mML-glutamine at 1×10⁵/well in 96-well flat-bottom plates. B cells werepretreated with serially diluted anti-human IL-21R antibodies in a 37°C. incubator adjusted to 5% CO₂ for 30 min. The treated B cells werethen stimulated with 0.5 μg/ml anti-CD40 mAb (BD Biosciences, San Jose,Calif.) and 10 ng/ml IL-21 cytokine for 3 days in a 37° C. incubatoradjusted to 5% CO₂. On day 3, cultures were pulsed with 0.5 μCi/well³H-thymidine (Perkin Elmer (NEN)) and harvested 5 hr later onto glassfiber filter mats. ³H-thymidine incorporation was determined by liquidscintillation counting. All of the improved antibodies neutralizedIL-21-dependent proliferation with greater potency than the parental18A5 antibody (FIGS. 12 a-b, Table 9; also see FIG. 26 e).

TABLE 9 Neutralization of Human Primary B cell ProliferationNeutralization of B cell Antibody proliferation IC₅₀ (nM) AbQ 0.16 AbR0.22 AbS 0.44 AbT 0.14 AbU 0.13 18A5 antibody 1.86

Example 9.10 Primary Human T cell Proliferation Assays

Anti-IL-21R antibodies were tested for their ability to inhibitIL-21-dependent proliferation of primary human CD4⁺ T cells. Buffy coatcells from healthy human donors were obtained from Massachusetts GeneralHospital. CD4⁺ T cells were isolated by negative selection usingROSETTESEP™ CD4⁺ T cell enrichment cocktail (StemCell Technologies),according to the manufacturer's instructions. The resulting populationwas ˜80-90% CD4⁺/CD3⁺ T cells. Enriched human CD4⁺ T cells wereactivated for 3 days with anti-CD3/anti-CD28-coated microspheres in RPMIcontaining 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine, and HEPES in a 37° C. incubator adjusted to 5% CO₂. Afteractivation, the microspheres were removed and the cells were washed andrested overnight at approximately 1×10⁶ cells/ml in culture medium. Therested cells were then washed again before addition to the assay plates.Serial dilutions of anti-human IL-21 receptor antibodies were made inculture medium in flat-bottomed 96-well plates, followed by thesequential addition of human IL-21 (20 ng/ml final concentration) andthe activated and rested CD4⁺ T cells (10⁵ cells/well). The plates werethen incubated for an additional 3 days and pulsed with 1 μCi/well³H-thymidine (Perkin Elmer (NEN)) during the final 6 hr of the assay.Cells were harvested onto glass fiber filter mats and ³H-thymidineincorporation was determined by liquid scintillation counting. All ofthe improved antibodies neutralized IL-21-dependent proliferation withgreater potency than the parental 18A5 antibody (FIG. 13, Table 10A;also see FIG. 26 f).

TABLE 10A Neutralization of Human Primary T cell ProliferationNeutralization of T cell Antibody Proliferation IC₅₀ (nM) AbO 0.06 AbP0.02 AbQ 0.08 AbR 0.04 AbS 0.06 AbT 0.03 AbU 0.03 18A5 antibody 1.42

Example 9.11 Primary Murine T cell Proliferation Assays

Anti-IL-21R antibodies were tested for their ability to inhibitIL-21-dependent proliferation of primary murine CD8⁺ T cells. Popliteal,axillary, brachial, and inguinal lymph nodes and spleens from12-week-old female BALB/C mice were collected. A single-cell suspensionof the spleen cells was depleted of red blood cells using 0.16 M NH₄Clin 0.017 M Tris (pH 7.4). The spleen and lymph node cells were pooledand enriched for CD8⁺ cells using a murine T cell CD8 Subset Column Kit(R&D Systems). Murine CD8⁺ cells (3×10⁴; suspended in DMEM containing10% fetal calf serum and supplemented with 0.05 mM (3-mercaptoethanol, 2mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamicin)were plated in 96-well, anti-mCD3 activation plates (BD Biosciences);mIL-21 (50 ng/ml) was added to all the wells. The test antibodies weretitered in triplicate beginning at 20 μg/ml. Cells were grown for 3 daysin a 37° C./10% CO₂ incubator. During the last 5 hr of culture, cellswere labeled with 0.5 μCi methyl-³H-thymidine/well (GE Healthcare). Thecells were harvested using a Mach III cell harvester (TomTec, Hamden,Conn.) and counted using a Trilux microbeta counter (Perkin Elmer).Aside from AbP, all of the improved antibodies neutralizedIL-21-dependent proliferation with greater potency than the parental18A5 antibody (FIG. 14, Table 10B; also see FIG. 26 g).

TABLE 10B Neutralization of Murine Primary T cell ProliferationNeutralization of T cell Antibody Proliferation IC₅₀ (nM) AbO 4.92 AbPno inhibition AbQ 0.85 AbR 0.13 AbS 0.02 AbT 0.61 AbU 1.79 18A5 antibody>85

Example 9.12 ADCC Assay

Anti-IL-21R antibodies were tested for their ability to induceantibody-dependent cellular cytotoxicity (ADCC) when bound to targetcells. The day before the experiment, PBMC were isolated from buffy coatby diluting the buffy coat 1:1 in PBS, layering it over FICOLL® (GEHealthcare) and centrifuging at 1200 g for 20 min. PBMCs were removedfrom the top of the FICOLL® layer, washed, and stimulated overnight with10 ng/ml IL-2 and 10 ng/ml IL-12 (R&D Systems). The day of theexperiment, stimulated PBMCs were collected by centrifugation andresuspended in media at 1×10⁸ cells/ml. BJAB cells were labeled with 0.5μM CFSE (MOLECULAR PROBES®, Invitrogen Corporation) for 10 min at 37°C., and then washed with fetal bovine serum once and PBS twice. Cellswere then plated into a 96-well flat-bottom plate at 2×10⁵ cells/well in100 μA media. Fifty μl of the 4× antibodies were added to the BJABcells, followed by 5×10⁶ PBMC in 50 μA, giving a final 1:25target:effector cell ratio. Cells were incubated at 37° C. for 6 hr andstained with propidium iodide (PI) to label dead and dying cells.Killing of target cells (CFSE⁺) was assessed by measuring PI staining ina FACSCALIBUR™ flow cytometer (BD Biosciences). Only one anti-IL-21Rantibody, AbZ, which has a wild-type human IgG1 constant region, showedADCC above the background level displayed by a control anti-IL-13antibody that did not bind to the target cells. All antibodies with thesame variable domains as AbZ, including forms with human IgG4 (AbY), andthose with double-mutant (AbX) and triple-mutant (AbT) forms of humanIgG1, showed only background levels of ADCC (FIG. 15). All otheranti-IL-21R antibodies tested contained the triple-mutant form of humanIgG1 and showed background ADCC. A positive control antibody, rituximab(RITUXAN®), induced ADCC in all experiments.

Example 9.13 C1q ELISA

In order to determine whether cell-surface binding by anti-IL-21Rantibodies is likely to lead to complement-dependent cytotoxicity (CDC),the antibodies were tested for their ability to bind to the complementcomponent C1q in an ELISA. IL-21R antibodies and rituximab (RITUXAN®)were diluted in PBS to 5 μg/ml. Diluted antibodies (100 μl) were coatedonto a COSTAR® high-binding ELISA plate (Corning Life Sciences, Lowell,Mass.) overnight at 4° C. Plates were washed 3× with PBS/Tween-20 andblocked with 200 μl of blocking buffer (0.1 M NaPO₄, 0.1 M NaCl, 0.1%gelatin, 0.01% Tween) for 1 hr at RT. Human serum previously determinedto contain C1q (Quidel, San Diego, Calif.) was diluted 1:50 in PBS.After 1 hr of blocking, plates were washed and 100 μl of diluted serumwas added to each well and incubated for 2 hr at RT on a shaker.Following three washes, 100 μl A of 0.1 μg/ml chicken polyclonalanti-human C1q antibody (AbCam, Cambridge, Mass.) was added to each welland incubated for 1 hr at RT. Plates were again washed and incubatedwith 100 μA of a rabbit polyclonal antibody to chicken Ig-Y—HRP diluted1:4000 (AbCam) for 1 hr at RT. Plates were washed and developed with TMBfor 5 min, followed by 50 μA of 1 M H₂SO₄ to stop the reaction, and thenread at 450 nm. Only one anti-IL-21R antibody, AbZ, which has awild-type human IgG1 constant region, showed C1q binding above thebackground level displayed by a control antibody with a triple-mutanthuman IgG1 constant region that had previously been shown to lack C1qbinding. All antibodies with the same variable domains as AbZ, includingforms with human IgG4 (AbY), and those with double-mutant (AbX) andtriple-mutant (AbT) forms of human IgG1, showed only background levelsof C1q binding (FIG. 16). All other anti-IL-21R antibodies testedcontained the triple-mutant form of human IgG1 and showed background C1qbinding.

Example 9.14 Cytokine Competition Assay

In order to demonstrate that antibody AbT binds to the murine IL-21R ina manner that competes with the IL-21 cytokine, a cytokine competitionassay was performed. Antibody AbT was coated at 1 μg/ml onto ELISAplates, which were then blocked with 1% BSA in PBS/0.05% Tween.Biotinylated murine IL-21R-His/FLAG (1.5 ng/ml) was added to the wells,either alone or in the presence of increasing concentrations of murineIL-21, and the binding of the receptor to the immobilized antibody wasdetected with HRP-conjugated streptavidin and subsequent incubation withTMB detection reagent. Mouse IL-21 was able to block the binding ofmIL-21R to AbT nearly completely above 4 ng/ml, indicating that theantibody and the cytokine compete for binding to murine IL-21R (FIG. 27a).

A second assay was performed to demonstrate that antibody AbS binds tothe murine IL-21R in a manner that competes with the IL-21 cytokineMurine IL-21R-Fc was captured on ELISA plates coated with an anti-mouseIgG2a antibody. Plates were blocked with 1% BSA in PBS and washed, andvarying concentrations of AbS were added to the plate in the presence of10 μg/m/mL-21. The binding of mIL-21 to the receptor was detected by anHRP-conjugated anti-His₆ antibody, and the binding of AbS to thereceptor was detected by an anti-human Ig antibody. Concentrations ofAbS above approximately 2 μg/ml completely prevented binding of mIL-21to mIL-21R-Fc, indicating that the antibody and the cytokine compete forbinding to murine IL-21R (FIG. 27 b).

Example 9.15 Inhibition of Rat T cell Proliferation by Anti-IL-21RAntibodies

Lewis female rat splenic T cells were purified to 95% CD3⁺ using Rat Tcell Enrichment Columns (RTCC-25; R&D Systems) according to themanufacturer's instructions. Serial dilutions of the anti-human IL-21Rantibodies and isotype control protein were made in culture medium(Dulbecco's Modified Eagle Medium containing 10% FCS, L-glutamine,beta-mercaptoethanol, nonessential amino acids, sodium pyruvate,penicillin, streptomycin, and gentamycin) in flat-bottomed 96-welltissue culture plates which had been precoated with 1 μg of anti-rat CD3antibody (BD Pharmingen Cat#554829), followed by the addition of 5 ng/mlrat IL-21 and 20,000 CD3 T cells per well. The cells were grown for 3days in a 10% CO₂, 37° C., humidified incubator. For the last 5 hr ofculture, cells were labeled with 0.5 μCi of ³H-thymidine (GE AmershamCat# TRA-120). The plates were harvested onto glass fiber filter mats bya Tomtec Mach III plate harvester and were counted on a Perkin Elmer1450 Microbeta Counter.

The response of the rat T cells to 5 ng/ml rat IL-21 was 6-fold abovethe background response to 1 μg of anti-CD3 alone. Antibodies AbS, AbU,AbV, and AbW were able to inhibit the ³H thymidine incorporationstimulated by 5 ng/ml rat IL-21 (57,000 cpm in the absence of antibodytreatment; FIG. 28). IC₅₀ values for neutralization in two independentexperiments are shown in Table 11.

TABLE 11 Blockade of IL-21-dependent Rat T cell Proliferation byAnti-IL-21R Antibodies. IC50 (nM) IC50 (nM) Antibody experiment 1experiment 2 AbS 35.98 27.07 AbU 172.79 105.46 AbV 70.55 59.23 AbW159.06 94.74

Example 9.16 Binding of AbS to Rabbit IL-21R

In order to demonstrate that antibody AbS binds to rabbit IL-21R, theextracellular domains of two isoforms of rabbit IL-21R were subcloned asFc fusions and transiently expressed in HEK293 cells. Rabbit IL-21R-Fc(either isoform 1 or isoform 2) was captured from conditioned mediumonto ELISA plates coated with anti-mouse IgG2a. Varying concentrationsof AbS were added, the plates were washed, and antibody binding wasdetected with an HRP-conjugated anti-human IgG antibody. AbS showedclear binding to both isoforms of rabbit IL-21R Fc (FIG. 29 a). Whenbinding of AbS to rabbit IL-21R-Fc was carried out in the presence of10% conditioned medium containing rabbit IL-21, binding of AbS to eitherreceptor isoform was reduced by approximately 10-fold, indicating thatAbS competes with rabbit IL-21 for binding to rabbit IL-21R (FIG. 29 b).

Example 10 Characterization of Improved IgG In Vivo Example 10.1Neutralization of IL-21R with the Anti-IL-21R Antibodies, AbS and AbT,Inhibits the Generation of IgG Antibody Responses to a T cell-dependentAntigen In Vivo

IL-21 is important for B cell isotype switching to certain subclasses ofIgG and differentiation to plasma cells. Thus, to determine the efficacyof the anti-IL-21R antibodies, AbS and AbT, in vivo, the ability ofthese antibodies to inhibit IgM and IgG antibody responses to the Tcell-dependent antigen, NP-chicken gamma globulin (NP-CGG) were testedin C57BL/6 mice. Mice were treated 3×/week with 10 mg/kg anti-IL-21Rantibody or isotype control beginning one day prior to immunization withNP-CGG. NP-specific IgG and IgM were detected by ELISA. NP-specific IgMand IgG antibodies were readily detected in serum of isotypecontrol-treated animals within 7 days following immunization, and theseresponses increased in magnitude to day 14 (FIG. 30 b). Treatment witheither AbS or AbT did not affect the magnitude of IgM responses in thisstudy. NP-specific IgG antibody responses were delayed in AbS- orAbT-treated cells, as demonstrated by a decreased response compared toisotype control on day 7. NP-specific IgG antibody responses weresimilar in isotype control-, AbS- and AbT-treated mice at day 14 (FIG.30 a). These data show that neutralization of IL-21R in vivo usingeither AbS or AbT can transiently inhibit the induction of early IgGantibody responses to a T cell-dependent antigen.

A second study testing AbS and AbT also yielded similar results (FIGS.30 c-f). NP-specific IgG responses were transiently reduced at day 7 ofimmunization in mice treated with either AbS or AbT, but were similar toisotype control-treated mice at later timepoints. Isotype-specificELISAs indicated that IgG2b and IgG2c were significantly reduced by AbSand AbT compared to controls at day 7 (FIGS. 30 e-f). These data showthat neutralization of IL-21R in vivo using either AbS and AbT cantransiently inhibit the induction of early IgG antibody responses to a Tcell-dependent antigen.

To test the effects of IL-21R neutralization on the maintenance ofestablished long-term humoral immunity, C57BL/6 mice were immunized withNP-CGG and rested for at least one month to allow them to generatememory B cells and long-lived plasma cells, which give rise to long-termIgG serum antibody titers. Approximately one month after immunization,mice were treated 3×/week i.p. with saline, or 10 mg/kg of either AbS orAbT, or isotype control antibody for two months. NP-specific IgG serumantibody titers were monitored every two weeks by ELISA. C57BL/6 micehad high titers of NP-specific IgG serum antibodies one month afterimmunization when compared to naïve C57BL/6 mice, consistent with theformation of long-term humoral immunity to NP. NP-specific IgG serumantibody titers remained stable over the course of the study in bothmice treated with the isotype control antibody, and treatment witheither AbS or AbT did not affect these antibody titers (FIG. 30 g).Following immunization, B cells generate plasma cells (in bone marrow),which give rise to long-term serum antibody titers. At the end of thestudy, the number of NP-specific IgG plasma cells in the bone marrow ofthe mice was measured, and the cell numbers were unaffected by treatmentwith AbS or AbT (FIG. 30 h). These data indicate that neutralization ofIL-21R with AbS and AbT in this treatment regimen does not affect themaintenance of established long-term serum antibody titers.

Example 10.2 Anti-IL-21R Antibody Function in a Model of SLE

The anti-IL-21R antibodies AbS and AbT were tested for their ability toameliorate disease in the MRL-Fas^(lpr) murine model of systemic lupuserythematosus (SLE). MRL-Fas^(lpr) mice spontaneously develop symptomsresembling those observed in human lupus, including high titers ofanti-double-stranded DNA (anti-dsDNA) autoantibodies in circulation,immunoglobulin and complement C3 deposits in the glomeruli, presence oflymphocytic infiltrates in the kidney and lung, and, in severe disease,proteinuria, lymphadenopathy, and skin lesions. Male MRL-Fas^(lpr) micewere obtained from Jackson Laboratory (Bar Harbor, Me.) and, starting at12 weeks of age, were given AbS, AbT, saline, or a control anti-humanIL-13 antibody with the same triple-mutant human IgG1 constant region ata dosage of 400 μg/mouse (10 mg/kg) 3×/week over 10 weeks via i.p.injection. Serum samples were taken biweekly and examined for anti-dsDNAantibodies by ELISA. Urine was collected biweekly and tested for protein(using protein test strips); neither control animals nor treated animalsdeveloped significant proteinuria. Animals were also monitored forenlarged lymph nodes and skin lesions; neither control animals nortreated animals showed abnormalities. After 10 weeks of treatment,animals were sacrificed, and kidney and brain sections were examined forIg and C3 deposits by immunohistochemistry. Cellular infiltrations intokidney and lungs were measured by examination of H/E-stained tissuesections.

Treatment of MRL-Fas^(lpr) mice with the IL-21R blocking antibody AbSsignificantly reduced anti-dsDNA IgG serum antibody levels as comparedto animals treated with saline or a control anti-human IL-13 antibody,beginning at 2 weeks post-treatment and continuing until 10 weekspost-treatment (FIG. 31). Anti-dsDNA antibodies were reduced toundetectable levels by AbS treatment in 8/10 mice at week 4 (FIG. 31 d),5/10 at week 6 (FIG. 31 e), and 7/10 at week 8 (FIG. 31 f), as comparedto saline and IL-13 control antibody-treated animals (all control micehad detectable anti-dsDNA antibody titers). Treatment with the IL-21Rblocking antibody AbT did not significantly affect anti-dsDNA IgG serumantibody levels.

FIG. 31 a shows anti-dsDNA antibody titers following treatment(AbS-treated group is significantly different from both saline- andanti-IL-13-treated groups (p<0.01)). FIG. 31 b shows prebleed anti-dsDNAantibody titers (saline-treated group is significantly different fromAbS-, AbT-, and anti-IL-13-treated groups (p<0.01)). The AbS-treatedgroup is significantly different from both saline- andanti-IL-13-treated groups (p<0.01) after 2 weeks of dosing (FIG. 31 c);4 weeks (FIG. 31 d); 6 weeks (FIG. 31 e); 8 weeks (FIG. 31 f); and 10weeks (FIG. 31 g).

Treatment with AbS also significantly reduced Ig and C3 immune complexdeposition and kidney pathology as compared to anti-IL-13antibody-treated controls (p<0.01) (FIGS. 32, 33). Twelve-week-old maleMRL-Fas^(lpr) mice were treated (10 mg/kg i.p., 3×/week) with eithersaline (control) or the indicated triple-mutant antibodies (ananti-human IL-13 human IgG1 A234 A235 A237 mutant antibody with noreactivity to murine IL-21 was used as an isotype control). Following 10weeks of treatment, mice were sacrificed and Ig and C3 deposits in thekidneys were identified by immunocytochemistry. FIG. 32 depicts IgGdeposits in kidneys of MRL-Fas^(lpr) mice treated as indicated (in FIG.32 b, glomeruli are indicated by dashed circles, and examples of diffusestain, indicating IgG deposits, are indicated with white arrowheads).Staining intensity was scored on a scale of 1-5 (FIG. 32 a). Kidney IgGdeposits in animals treated with AbS were significantly lower (p<0.01)than those in IL-13 triple-mutant-treated controls. FIG. 33 depicts IgM(FIG. 33 a) and complement C3 (FIG. 33 b) deposits in kidneys ofMRL-Fas^(lpr) mice treated as indicated. Staining intensity was scoredon a scale of 1-5. IgM and complement C3 deposition in the kidneys weresignificantly reduced (p<0.05) by AbS compared to the anti-IL-13 controlantibody (FIG. 33). FIG. 34 depicts deposition of IgG (FIG. 34 a), IgM(FIG. 34 b), and C3 (FIG. 34 c) in the brains of treated mice. IgG(p<0.05; FIG. 34 a) but not IgM (FIG. 34 b) or C3 (FIG. 34 c) depositswere reduced in the brains of AbS-treated mice as compared to anti-IL-13antibody-treated controls. Staining intensity was scored on a scale of1-5. Treatment with AbT did not reduce immune complex deposition in thekidneys or brains of MRL-Fas^(lpr) mice (FIGS. 32-34).

Infiltration of lymphocytes into the kidneys and lungs of MRL-Fas^(lpr)mice was also examined histologically. Twelve-week-old maleMRL-Fas^(lpr) mice were treated (10 mg/kg i.p., 3×/week) with eithersaline (control) or the indicated triple-mutant antibodies. Following 10weeks of treatment, mice were sacrificed and H/E-stained kidney and lungsections were examined for lymphocyte infiltration. Lymphocyte numberswere scored on a scale of 1-5. In the kidney, AbS but not AbTsignificantly reduced lymphocyte infiltration in three zones:cortex-interstitium (a support structure for the glomeruli) (FIG. 35 a),cortex-perivascular region (FIG. 35 b), and peripelvic region (near theorigin of the ureter) (FIG. 35 c) (p<0.01). Both AbS and AbT treatmentsignificantly reduced the number of lymphocytes measured in the lungs ofMRL-Fas^(lpr) mice as compared to saline-treated and anti-IL-13antibody-treated controls (p<0.01 and p<0.05, respectively; FIG. 36).

Taken together, these data show that treatment with AbS ameliorateslupus-like disease in MRL-Fas^(lpr) mice, whereas treatment with AbTseemed to be less efficacious in this model of SLE. To examine thediscrepancy in efficacy of AbS and AbT treatment of MRL-Fas^(lpr) mice,sera from these mice were examined for anti-product antibody responsesby ELISA. Twelve-week-old male MRL-Fas^(lpr) mice were treated (10 mg/kgi.p.; 3×/week) with the indicated triple-mutant antibodies. Serumcollected biweekly was tested by ELISA for the presence of murineantibodies capable of binding to the same human antibodies with whichthe mice were treated. MRL-Fas^(lpr) mice generated anti-human antibody(MAHA) responses to all three tested antibodies as early as 2 weeksafter treatment (FIG. 37 a). However, anti-product IgG antibodyresponses were more than 10-fold greater against AbT or the controlantibody than against AbS (p<0.05 for AbS vs. IL-13 control). It isunlikely that the AbS CDR sequence is more immunogenic than that of AbT,as for both AbS and AbT the majority of anti-product antibodiesrecognized epitopes common to both molecules (FIG. 37 b)

To examine the dose-dependency of IL-21R neutralization on diseasedevelopment in the MRL-Fas^(lpr) model, eight-week-old female mice wereadministered either AbS (10, 5 or 2.5 mg/kg doses), AbT (20 mg/kg doses)or isotype control antibody (20, 10, 5, or 2.5 mg/kg doses) i.p. 3×/weekfor 10 weeks. Mice were tested for anti-dsDNA serum antibodies, andexamined for proteinuria, lymphadenopathy, and skin lesions every twoweeks. After 10 weeks of dosing, animals were sacrificed and kidneysections were examined for immunoglobulin deposits byimmunohistochemistry, and immune cell infiltrates were measured byexamination of H&E-stained kidney sections.

MRL-Fas^(lpr) mice had detectable levels of anti-dsDNA IgG antibodies atthe onset of the study, and these antibodies increased in titer over thecourse of the study in all treatment groups. However, treatment with AbSat all doses tested (10, 5 and 2.5 mg/kg) significantly reduced antibodytiters in MRL-Fas^(lpr) mice in a dose-dependant fashion when comparedto isotype control-treated mouse serum (FIG. 38 a). AbT (20 mg/kg) alsoreduced anti-dsDNA antibody titers in these mice, but only at one timepoint (week 2 of dosing) (FIG. 38 b).

To further assess the impact of IL-21R neutralization on diseaseprogression in MRL-Fas^(lpr) mice, the mice were examined for clinicalsigns of disease. Very few of the MRL-Fas^(lpr) mice used in this studydeveloped skin lesions or clinically significant proteinuria, so theeffects of AbS and AbT treatment on these aspects of disease could notbe assessed. Treatment with AbS and AbT at all of the doses tested didnot affect the development of lymphadenopathy in the study. However, IgGdeposits and immune cell infiltrates were readily observed in kidneysfrom isotype control-treated mice, consistent with the development oflupus nephritis in these mice. Treatment with 10 mg/kg AbS significantlyreduced the mean number of immune cell infiltrates and IgG deposits inthe kidney, whereas treatment with 5 and 2.5 mg/kg AbS had no effect onthese parameters in this study (FIG. 38 c-d). Treatment with AbT at the20 mg/kg dose increased mean number of immune cell infiltrates and didnot affect IgG deposits in the kidney of MRL-Fas^(lpr) mice in thisstudy (FIG. 38 c-d).

Example 10.3 IL-21R Antibodies Block Cellular Infiltration in a MurineAir Pouch Assay

The ability of AbS and AbT to neutralize IL-21R function in vivo wastested in a short-term murine air pouch assay. Air pouches were createdby injecting 8-10 week old BALB/C mice with 3 ml of air under the dorsalskin. Three days later, pouches were reinflated. Two days afterreinflation, either saline (control) or the indicated antibodies wereinjected i.p. at the indicated dose. Murine IL-21 (100 ng) was injectedinto the air pouch 24 hr after antibody injection. Six hr later, thepouches were washed out with 3 ml PBS, and total cell counts (FIG. 39a), monocytes (FIG. 39 b), lymphocytes (FIG. 39 c), and neutrophils(FIG. 39 d) were determined with a CELL-DYN® (Abbott, Abbott Park,Ill.).

Injection of murine IL-21 into the air pouch led to a significantincrease in total cells (p=0.0002), monocytes (p=0.0137), lymphocytes(p=0.0035), and neutrophils (p=0.0004) in the air pouch 6 hr later ascompared to saline (FIG. 39 a). Treatment with either AbS or AbT led tosignificant reductions in cellular infiltration into the air pouch ascompared to treatment with the control IgG1 (anti-IL-13). Total cellularinfiltration was reduced significantly compared to the control IgG byboth AbS (10 mg/kg, p=0.0031; 1 mg/kg, p=0.00426) and AbT (10 mg/kg,p=0.0198) (FIG. 39 a). Monocyte infiltration was significantlydecreased: AbS (5 mg/kg, p=0.0031; 2 mg/kg p=0.0239; 1 mg/kg, p=0.0008)and AbT (10 mg/kg, p=0.0002; 5 mg/kg p=0.0066; 1 mg/kg, p=0.0009) (FIG.39 b). Lymphocyte infiltration was also significantly decreased: AbS (1mg/kg, p=0.0049) and AbT (10 mg/kg, p=0.0222) (FIG. 39 c). Neutrophilinfiltration was significantly reduced by AbS (10 mg/kg, p=0.0032), butnot AbT.

Similar results were obtained in a second study conducted to examine theability of AbS and AbT in the murine air pouch model. Total cellinfiltration into the air pouch was significantly reduced by AbS whenadministered at 10 (p=0.0113), 5 (p=0.0027), and 2 mg/kg (p=0.029), andby AbT at 10 mg/kg (p=0.0007) (FIG. 39 e).

The effect of AbS on cell infiltration in response to IL-21 in the ratair pouch model was also examined to determine if these antibodies couldneutralize IL-21R in the rat. Pouches were created by injecting8-week-old female S-D rats with 20 ml of air into the subcutaneoustissue of the back. Three days later, pouches were reinflated byinjecting an additional 10 mL of air into the pouch. Two days later, therats were injected with the designated amount of isotype control or AbSantibody. The next day, either saline or 1-20 μg of murine IL-21 wasinjected into the pouch, and six hr later the pouch contents were washedout and total cells counted using a Cell Dyne machine.

Injection of 1 μg of murine IL-21 into the air pouch did not result insignificant cell infiltration into the pouch. However, administration of20 μg of murine IL-21 led to an increase in total cells in the air pouch6 hr later as compared to saline, and treatment with 10 mg/kg AbSsignificantly reduced IL-21 mediated cell infiltration into the pouchunder these conditions (p<0.05) (FIG. 39 f). To determine the minimaldose of AbS that could inhibit cell infiltration into the air pouch inthis rat model, additional studies were performed in which rats wereadministered 20 μg of murine IL-21 and either 10 mg/kg isotype controlor 10, 3 or 1 mg/kg of AbS. Administration of 10 mg/kg AbS significantlyreduced cell infiltration into the rat air pouch (p<0.05). Anonsignificant increase in cell infiltration into the air pouch wasobserved in rats treated with 3 mg/kg of AbS. Treatment with 1 mg/kg AbSsignificantly increased cell infiltration into the pouch compared toisotype control treated rats (p<0.05) (FIG. 39 g). These observationswere repeated in a second study, in which treatment with 10 mg/kgsignificantly reduced cell infiltration into the rat air pouch inducedby 20 μg murine IL-21 (p<0.05); in this study, treatment with both 3 and1 mg/kg AbS significantly increased cell infiltration into the pouch(p<0.05) (FIG. 39 h).

Treatment with AbS significantly increased cell infiltration into thepouch in response to IL-21 when AbS was administered at low doses (e.g.,1 mg/kg). To determine if administration of antibody alone into thepouch at low concentrations (1 mg/kg) elicited cell migration into thepouch, an additional study was performed in which rats were treated with1 mg/kg AbS or isotype control antibody in the absence of IL-21treatment. As observed in previous studies, administration of AbS at 10mg/kg significantly reduced IL-21-driven cell infiltration into thepouch (p=0.0075), whereas administration of AbS at 1 mg/kg significantlyincreased IL-21-induced cell infiltration into the pouch (p=0.0257).Treatment with either AbS or isotype control antibody at 1 mg/kg in theabsence of IL-21 treatment did not increase cell infiltration into thepouch, indicating that the increased cell infiltration into the pouchelicited by treatment with AbS at 1 mg/kg was dependent onadministration of both murine IL-21 and anti-IL-21R antibody in thismodel (FIG. 39 i).

To determine whether the increase in cells in the rat air pouch modelwas dependent on administration of the dose of murine IL-21 (20 μg), anexperiment was done in which rats were treated with either 10, 20 or 40μg murine IL-21 and either 1 or 10 mg/kg AbS. A significant inhibitionof cell infiltration into the pouch was observed with 10 mg/kg AbS inresponse to both 10 (p=0.003) and 20 μg (p=0.003) murine IL-21.Treatment with 10 mg/kg AbS did not affect cell infiltration in responseto 40 μg murine IL-21 in this study. Treatment with 1 mg/kg AbSsignificantly increased cell infiltration into the air pouch in responseto 20 μg murine IL-21 (p=0.0454), as previously observed. However,treatment with 1 mg/kg AbS did not affect cell infiltration into rat airpouch in response to either 10 or 40 μg murine IL-21, indicating thatincreased cell infiltration into the rat air pouch is very specific tothe dose of 1 mg/kg Abs and 20 μg murine IL-21 in this model (FIG. 39j). These data indicate that AbS can affect cell infiltration into therat air pouch in response to murine IL-21, with inhibition of thisresponse achieved with a dose of 10 mg/kg of AbS.

Example 10.4

Pharmacokinetics in CD-1 Mice After Intravenous or SubcutaneousAdministration of AbS

The serum concentrations of human anti-IL-21R antibodies were determinedby qualified ELISAs as described in Table 13. The anti-IL-21R ELISA useda monomeric His-tagged IL-21R as a capture reagent and an anti-human-Fc(conjugated to horseradish peroxidase (HRP)) as a detector reagent. Theenzyme substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), was used toproduce a colored endproduct to visualize the bound test article.Optical density (OD) was measured colorimetrically at a wavelength of405 or 450 nm. Sample concentrations were determined by interpolationfrom a standard curve that was fit using a four-parameter equation.

Pharmacokinetic parameters were calculated based on mean concentrations.Individual concentration values below the LOQ were treated as zero forcalculation of the mean and SD. The PK parameters were determined usinga noncompartmental analysis module (Model 200 for i.p. and s.c. dosingand Module 201 for i.v. dosing) of the PK software package WinNonlin(version 4.1; Pharsight, Mountain View, Calif.). The program applies amodel-independent approach and the standard methods described by Gibaldiand Perrier (Pharmacokinetics (2nd ed. 1982) Marcel-Dekker, Inc., NewYork). The area under the serum concentration vs. time curve (AUC) wascalculated using the linear trapezoidal method. The slope of theapparent terminal phase was estimated by log-linear regression using atleast three data points and the terminal rate constant (λ) was derivedfrom the slope. AUC_(0-∞) was estimated as the sum of the AUC_(0-t)(where t is the time of the last measurable concentration) and C_(t)/λ.The apparent elimination half-life (t_(1/2)) was calculated as 0.693/λ.Predictions of concentrations after a multiple dose regimens, wereconducted by nonparametric superposition using WinNonlin software.

After intravenous administration of 10 mg/kg of AbS to male CD-1 mice,the exposure (AUC_(0-∞)) of AbS was 7272 μg*hr/ml. The meanconcentration at the first sampling time point after intravenousadministration (C_(5min)) was 113 μg/ml. The elimination of AbS in CD-1mice was relatively slow, as evidenced by the low total body clearance(CL) of ˜1.4 ml/hr/kg and long elimination half-life (t_(1/2)) of 162 hr(˜6.8 days). The steady-state volume of distribution (Vd_(SS)) was 306ml/kg, suggesting that AbS was mainly confined to the vascular system(FIG. 40; Table 14).

Pharmacokinetics of AbS in CD-1 mice appeared linear in the 10-100 mg/kgdose range. After intravenous administration of 100 mg/kg of AbS to maleCD-1 mice, AUC_(0-∞) was 75792 μg*hr/ml, C_(5min) was 1160 μg/ml, CL was˜1.3 ml/hr/kg, Vd_(SS) was 473 ml/kg, and elimination half-life was ˜391hr (˜16.2 days) (FIG. 40; Table 14).

After 10 mg/kg s.c. administration of AbS to CD-1 mice the absorption ofAbS was slow (T_(max) of 48 hr) and the subcutaneous bioavailability was81%. The mean t_(1/2) value after subcutaneous administration was ˜195hr (˜8.1 days) and similar to that observed after 10 mg/kg intravenousadministration.

The presence of anti-AbS antibodies was evaluated using a qualifiedimmunoassay at 576 hr (24 days) and 672 hr (28 days) following 10 mg/kgi.v. or s.c. administration of AbS to CD-1 mice (n=8 per time point pergroup). An electrochemiluminescent, paramagnetic bead assay was used todetect anti-AbS antibodies. In this assay, samples were coincubated withbiotinylated AbS overnight. Streptavidin-coated paramagnetic beads wereincubated with the mixture. After incubation with the beads, the platewas placed in the BioVeris M-Series 384 Analyzer. A magnet was appliedto capture the paramagnetic beads onto a surface electrode, and unboundreactants were washed away. The ruthenylated AbS captured on the beadswas electrically excited by a voltage application, resulting in theproduction of light. The light was measured by photodetectors with theread-out in response units (RU).

Positive and negative controls were also used to determine the cutpointRU, which was defined as twice the mean RU of the negative control.Samples were initially tested in a screening format at dilutions of 1:25and 1:75. Samples generating an RU greater than or equal to the cutpointRU were considered positive and reanalyzed in a full-dilution series toconfirm the positive result and determine the titer (the reciprocaldilution that would generate an RU equal to the cutpoint RU). Forpositive samples, the log of titer is reported. The minimum requireddilution was 1:25, and the limit of detection was 1.40 (the log of 25).Therefore, negative samples were designated as <1.40.

In both i.v. and s.c. treatment groups, 5 or 6 (of 8) mice per timepoint per group tested positive for anti-AbS antibodies (Table 15). Themajority of the mice with detectable anti-AbS antibodies had lower AbSconcentrations than those observed in animals without anti-AbSantibodies; of some note, the high levels of AbS in some samples couldhave interfered with the detection of anti-AbS antibodies. The summaryof the onset of anti-AbS antibody response across various times, andacross animal species and strains/models, is shown in Table 23.

The presence of anti-AbS antibodies was also tested at 648 hr (27 days),984 hr (41 days), and 1320 hr (55 days) following 100 mg/kg i.v. of AbSto CD-1 mice (n=3 per time point). None of the mice tested were positivefor anti-AbS antibodies at these time points.

Example 10.5 Pharmacokinetics in DBA and MRL-Fas^(lpr) Mice AfterIntraperitoneal Administration of AbS

The serum concentrations of human anti-IL-21R antibodies were determinedby qualified ELISAs as described in Table 13. The anti-IL-21R ELISA usedanti-human-Fc as a capture reagent and biotinylated anti-human-Fc as adetector reagent. Avidin-HRP, and the enzyme substrate TMB or 2,2′-azinodi(3-ethyl-benzthiazoline-6-sulfonate) (ABTS), were used to produce acolored endproduct. OD was measured colorimetrically at a wavelength of405 or 450 nm. Sample concentrations were determined by interpolationfrom a standard curve that was fit using a four-parameter equation. PKparameters were calculated as noted in Example 10.4. Predictions ofconcentrations after a multiple dose regimens, were conducted bynonparametric superposition using WinNonlin software and assuming linearkinetics in MRL-Fas^(lpr) and DBA mice in the 2.5-10 mg/kg dose range(single dose).

Following a single i.p. dose of 8 mg/kg of AbS to male DBA mice, themaximum serum concentration (C_(max)) and exposure (AUC_(0-∞)) of AbSwere 62 μg/ml and 7229 μg*hr/ml, respectively. The T_(max) and theelimination half-life (t_(1/2)) of AbS were 6 hr and 140 hr (˜5.8 days),respectively (FIG. 41). Out of eight animals tested, three developedanti-AbS antibody response at 672 hr, determined using the paramagneticbead assay described in Example 10.4 (Table 12). The summary of theonset of anti-AbS antibody across various times and species/strains isshown in Table 23.

TABLE 12 Formation of Anti-AbS Antibodies After a Single 8 mg/kg i.p.Dose to DBA Mice Onset (hr) 408 hr 504 hr 576 hr 672 hr Positive 0 of 80 of 8 0 of 8 3 of 8

Following a single intraperitoneal dose of 10 mg/kg to female,12-week-old MRL-Fas^(lpr) mice, the maximum serum concentration(C_(max)) and exposure (AUC_(0-∞)) of AbS were 51 μg/ml and 2798μg*hr/ml, respectively. The T_(max) and the elimination half-life(t_(1/2)) of AbS were 3 hr and 46 hr (˜1.9 days), respectively (FIG.41).

Compared to DBA and CD-1 mice, dose-normalized exposure of AbS appearedto be lower in MRL-Fas^(lpr) mice (Table 14). This was not entirelyunexpected, as fast elimination of normal IgG in MRL-Fas^(lpr) mice(especially with disease symptoms) and in SLE patients has been reported(see Zhou et al. (2005) Lupus 4(6):458-66; Newkirk et al. (1996) Clin.Exp. Immunol. 106(2):259-64; Wochner (1970) J. Clin. Invest.49(3):454-64). The hypercatabolism of mouse IgG in MRL-Fas^(lpr) micehas been proposed to be due to the disease-induced impairment of thefunction of the receptor FcRn, which regulates the homeostasis of IgG(Zhou et al. (2005) supra). As AbS is a human IgG, differences inelimination of AbS at the terminal phase among the different strains ofmice also may be explained, at least in part, by a differential MAHAresponse.

When AbS was administered to MRL-Fas^(lpr) mice i.p. at 2.5, 5, or 10mg/kg per dose 3×/week for 10 weeks (Example 10.2), all dose groups hadsignificant reduction in titers of anti-dsDNA antibodies compared to theisotype control (anti-human IL-13 human IgG1 triple-mutant). For the 2.5and 10 mg/kg dose groups, steady-state trough levels of AbS were assayedby ELISA. At 2- and 4-week time points, almost all samples tested fromthe 2.5 mg/kg group had undetectable levels (<34 ng/ml) of AbS in theserum; one sample had very low levels (57 ng/ml). For the 10 mg/kggroup, there was high interanimal variability in the AbS serumconcentrations; however, median steady-state trough levels were ˜3-10fold higher, compared to those predicted by simulations using PK datafrom the single dose study in MRL-Fas^(lpr) mice (FIG. 42). Whensingle-dose i.p. data obtained in the DBA mouse strain were used forpredictions, there appeared to be an improved correlation between theobserved and predicted AbS concentrations in the 10 mg/kg/dose group(FIG. 42). Without intending to be bound by theory, a possibleexplanation for the undetectable levels of AbS in the low-dose group ismarked MAHA production triggered by multiple administration of AbS atrelatively low dose levels, as week-2 serum samples from the 2.5mg/kg/dose group were assayed for anti-AbS antibodies and all sampleshad very high titers above the upper limit of quantitation of 4.74 logtiter units. However, in lieu of expected pharmacological action of AbSto delay and/or reduce IgG responses, it is possible that higher dosageof AbS under the multiple dose regimens would reduce MAHA responseagainst itself, resulting in higher observed serum AbS concentrations,compared to those expected based solely on the single-dose PK data. Infact, in MRL-Fas^(lpr) mice, MAHA response to AbS was ˜10-fold lower,compared to that to an isotype control human IgG administered via thesame multiple dose regimen of 10 mg/kg 3×/week for 10 weeks (FIG. 37 a).

Example 10.6 PK of AbS in Female Cynomolgus Monkeys

PK of AbS in female monkeys were determined following a single 10 mg/kgi.v. or s.c. administration. Individual animal and meanconcentration-time profiles from this study are shown in FIGS. 43 a and43 b, respectively, and mean PK parameters are summarized in Table 14.

The serum concentrations of human anti-IL-21R antibodies were determinedby qualified ELISAs as described in Table 12. The anti-IL-21R ELISA useda monomeric His-tagged IL-21R as a capture reagent and an anti-human-Fcconjugated to HRP as a detector reagent, as described for CD-1 mice inExample 10.4. The serum concentration of the isotype control antibody inmonkeys (anti-IL-13 antibody) was also measured by ELISA. In this assay,the recombinant human IL-13 ligand, which contains a FLAG octapeptidefusion tag, was captured by an anti-FLAG monoclonal antibody. The serumsamples containing anti-IL-13 antibody were detected with ananti-human-Fc-HRP. The enzyme substrate ABTS was used to produce acolored endproduct to visualize the bound test article. PK parameterswere calculated for each individual animal using noncompartmentalmethods.

All six female monkeys that were administered AbS at 10 mg/kg (n=3 forboth i.v. and s.c. routes of administration) had a sharp drop in AbSlevels in the terminal phase (at ˜408 hr post-dose), suggesting thepossible formation of anti-product antibodies. The elimination half-lifevalues in the 10 mg/kg groups were calculated with and without the timepoints at which a sharp concentration drop was observed.

After a single 10 mg/kg i.v. administration of AbS to female cynomolgusmonkeys, the mean serum clearance (CL) was 1.03 ml/hr/kg and the meanelimination half-life (t_(1/2)) was 74 hr (˜3 days). When time points atwhich a sharp concentration drop was observed were excluded fromcalculations, the mean t_(1/2) estimate was ˜7.9 days. The meansteady-state volume of distribution (Vd_(SS)) was low (˜125 ml/kg),suggesting that AbS was mainly confined to the vascular system. The meanexposure (AUC_(0-∞)) of AbS was 9728 μg*hr/ml.

After a single 100 mg/kg i.v. administration of AbS to cynomolgusmonkeys, CL was ˜1.08 ml/hr/kg, t_(1/2) was 279 hr (˜11.6 days),AUC_(0-∞) was ˜92867 μg*hr/ml, and Vd_(SS) was ˜211 ml/kg. Thus, ingeneral, the PK of AbS were approximately linear in the 10-100 mg/kgdose range.

After 10 mg/kg s.c. administration of AbS to female monkeys, theabsorption of AbS was slow (mean T_(max) of ˜34 hr) and the mean s.c.bioavailability was ˜43%. The mean t_(1/2) value after 10 mg/kg s.c.administration to monkeys was 52 hr (˜2.2 days), and similar to thatobserved after 10 mg/kg i.v. administration. When time points at which asharp concentration drop was observed were excluded from calculations,the mean t_(1/2) was 258 hr (˜10.8 days).

All six female monkeys that were administered AbS at 10 mg/kg haddetectable anti-AbS antibodies starting at 504 hr (3 weeks) post-dose(Table 16), determined using the paramagnetic bead assay described inExample 10.4. These data are consistent with the observed sharp drop inAbS serum levels. The anti-AbS response persisted at all subsequent timepoints (up to 27 weeks). Thus, the anti-AbS antibody response appears toaffect the PK of AbS in monkeys after a single 10 mg/kg i.v. or s.c.administration. For the 100 mg/kg i.v. dose group, 1 of 3 monkeys waspositive for anti-AbS antibodies at weeks 6 and 8, with log titers of1.8 and 4.6, respectively.

TABLE 13 ELISA Summary for AbS in Mouse and Female Monkey Serum Range ofSerum quantitation Minimum Standard used for Species Assay Assay (ng/mLin Required curve (ng/ml assay strain Capture Detector 100% serum)Dilution in 5% serum) diluent Mouse anti-human anti-human 32-500 200.78-100  Sprague- DBA IgG IgG-biotin Dawley rat Mouse anti-humananti-human 33.4-630   20 0.514-102   C57BL/6 MRL^(lpr) IgG IgG-biotinmouse Mouse Monomeric anti-human 45-768 20   1-38.4 CD-1 CD-1 His-taggedIgG-HRP mouse Il-21R Monkey Monomeric anti-human 30-512 20 0.44-38.4Pooled (cyno) His-tagged IgG-HRP monkey Il-21R

TABLE 14 Mean PK Parameters of AbS in Mice and Female Monkeys Dose(mg/kg), C_(5 min) or Species Route, C_(max) ^(a) T_(max) AUC_(0-∞) CLVd_(ss) t_(1/2) F strain Protocol (μg/ml) (hr) (μg * hr/ml) (ml/hr/kg)(ml/kg) (hr) (%) Mouse 8, ip 62 6 7229 NA NA 140 ND DBA Mouse 10, ip 513 2798 NA NA  46 ND MRL^(lpr) 2.5, ip 12 3 823 NA NA 12 (54)^(b) NDMouse 10, iv 113 NA 7272 1.38 306 162 NA CD-1 10, sc 17 48  5913 NA NA195 81 100, iv 1160 NA 75792 1.32 473 391 NA Mouse 2.5, iv 29 NA 19271.30 208 120 NA C57BL/6 Mouse 2.5, iv 32 NA 3415 0.73 273 256 NA IL-21Rknockout Monkey 10, iv 177 ± 10 NA 9728 ± 621 1.03 ± 0.07 125 ± 16 74 ±46 NA (cyno) (190 ± 38)^(b ) 10, sc 15 ± 3 34 ± 34 4188 ± 542 NA NA 52 ±20 43 ± 6 (258 ± 35)^(b ) 100, iv 2030 ± 95  NA 92867 ± 9768 1.08 ± 0.11211 ± 11 279 ± 28  NA ^(a)C_(5 min) was determined for i.v. route, orC_(max) was determined for i.p. and s.c. routes. ^(b)Terminal half-lifewas calculated excluding the data points with the sharp concentrationdrop. NA = not applicable ND = not determined

TABLE 15 Formation of Anti-AbS Antibodies (Log Titer) in Male CD-1 MiceAfter a Single IV or SC Dose of 10 mg/kg Time (hr) SAN 576 672Intravenous Group 26 2.65 27 2.54 28 <1.40^(a) 29 2.16 30 <1.40^(a) 402.81 41 2.34 42 2.32 43 2.79 44 <1.40^(a) 45 3.25 46 1.79 47 3.28 48<1.40^(a) 49 3.21 50 3.18 Subcutaneous Group 76 2.30 77 2.55 78<1.40^(a) 79 2.71 80 1.87 90 <1.40^(a) 91 3.75 92 2.35 93 <1.40^(a) 942.75 95 <1.40^(a) 96 3.27 97 3.53 98 3.73 99 <1.40^(a) 100 3.65^(a)<1.40 signifies a negative result. SAN = study animal number

TABLE 16 Formation of Anti-AbS Antibodies (Log Titer) in FemaleCynomolgus Monkeys After a Single IV or SC Dose of 10 mg/kg IntravenousGroup Time (hr) SAN 1 SAN 2 SAN 3  336 <1.40^(a) <1.40 <1.40  504 2.352.33 2.26  672 3.61 3.22 2.47  804 3.78 3.61 3.31 1008 3.80 3.72 4.181176 3.72 3.76 4.09 2016 3.70 4.44 4.14 2856 3.60 4.67 4.44 3696 3.604.71 4.24 4536 3.53 4.64 4.31 Subcutaneous Group Time (hr) SAN 4 SAN 5SAN 6  336 <1.40^(a) <1.40 <1.40  504 3.00 2.68 2.39  672 3.52 3.64 2.59 804 4.10 3.71 2.47 1008 3.91 3.73 2.89 1176 4.11 3.59 3.27 2016 4.293.77 3.65 2856 4.25 3.61 3.50 3696 4.26 3.49 3.45 4536 4.39 3.56 3.58^(a)<1.40 signifies a negative result. SAN = study animal number

Example 10.7 PK of AbT in Mice

The serum concentrations of human anti-IL-21R antibodies were determinedby qualified ELISAs as described in Table 17. The anti-IL-21R ELISA useda monomeric His-tagged IL-21R as a capture reagent and an anti-human-Fc(conjugated to horseradish peroxidase (HRP)) as a detector reagent forCD-1 mice, and anti-human Fc as a capture reagent and biotinylatedanti-human Fc as a detector reagent for DBA and MRL-Fas^(lpr) mice. PKparameters were calculated as noted in Example 10.4. Predictions ofconcentrations after a multiple dose regimens were conducted bynonparametric superposition using WinNonlin software and assuming linearkinetics in MRL-Fas^(lpr) and DBA mice in the 2.5-10 mg/kg dose range(single dose) for AbS and in the 8-20 mg/kg dose range (single dose) forAbT.

After i.v. administration of 10 mg/kg of AbT to male CD-1 mice, theexposure (AUC_(0-∞)) of AbT was 4551 μg*hr/ml (FIG. 44 a; Table 18). Themean concentration at the first sampling time point after i.v.administration (C_(5min)) was ˜70 μg/ml. The elimination of AbT in CD-1mice was relatively slow, as evidenced by the low total body clearance(CL) of ˜2.2 ml/hr/kg and long elimination half-life (t_(1/2)) of 210 hr(˜8.8 days). The steady-state volume of distribution (Vd_(SS)) was 572ml/kg. Following 10 mg/kg i.v. administration to mice, AbT appeared tohave higher CL (˜60% increase), lower AUC_(0-∞) (˜37% decrease) andlarger Vd_(SS) (˜87% increase), compared to the corresponding values forAbS.

Following a single i.p. dose of 8 mg/kg to male DBA mice, the maximumserum concentration (C_(max)) and exposure (AUC_(0-∞)) of AbT were 21μg/ml and 3320 μg*hr/ml, respectively (FIG. 44 c; Table 18). The T_(max)and the elimination half-life (t_(1/2)) of AbT were 1 hr and 80 hr (˜3.3days), respectively. After i.p. administration to DBA mice, AbT exposureand half-life were reduced ˜54% and ˜43%, respectively, compared to thecorresponding PK parameters of AbS.

Following a single i.p. dose of 10 mg/kg to female 12-week-oldMRL-Fas^(lpr) mice, the maximum serum concentration (C_(max)) andexposure (AUC_(0-∞)) of AbT were 23 μg/ml and 957 μg*hr/ml, respectively(FIG. 44 b; Table 18). The T_(max) and the elimination half-life(t_(1/2)) of AbT were 6 hr and 21 hr, respectively. After i.p.administration to MRL-Fas^(lpr) mice, AbT exposure and half-life wasreduced ˜66% and ˜54%, respectively, compared to the corresponding PKparameters of AbS. At 2 weeks, the only time point for AbT at which areduction in anti-ds DNA titers was observed, median AbT levels were4-5-fold higher compared to those predicted by simulations using PK datafrom the single-dose study in MRL-Fas^(lpr) mice and similar compared tothose predicted by simulation with single-dose data obtained from DBAmice (FIG. 45). This observation is in line with that for the AbS 10mg/kg group. Without intending to be bound by theory, a possibleexplanation for relatively lower levels of AbT at the 2-week time point(compared to those of AbS) and undetectable levels of AbT at later timepoints is marked MAHA production triggered by multiple administrationsof AbT and the inability of AbT to suppress the MAHA response againstitself.

In line with results described for AbT, dose-normalized exposure of AbTappeared to be lower in MRL-Fas^(lpr) mice, compared to those in DBA andCD-1 mice (Table 18).

Unlike AbS, when AbT was administered to MRL-Fas^(lpr) mice i.p. at 10mg/kg or 20 mg/kg per dose 3×/week for 10 weeks, only limited effects onthe development of disease in MRL-Fas^(lpr) mice were observed (Example10.2). Administration of AbT (10 mg/kg) did not result in significantreduction of titers of anti-dsDNA at all time points evaluated. Twentymg/kg administration of AbT resulted in transient reduction in titers ofanti-dsDNA at 2 weeks; however anti-dsDNA titers were not different fromcontrol at later time points (4, 6, 8, and 10 weeks) (Table 19). For the20 mg/kg AbT group, steady-state trough levels of AbT were assayed byELISA. There was high interanimal variability in the AbT serumconcentrations; however, in general, median steady-state trough levelsof AbT appeared to have an inverse correlation with the effect on mediananti-dsDNA titers (Table 19). At 2 weeks, median AbT levels were ˜8-foldhigher than those at 4 weeks (but still lower than median AbS levels at2 weeks in the 10 mg/kg group). At later time points (4, 6, 8, and 10weeks), median AbT trough levels were less than the limit of detection(˜66 ng/ml). At 2 weeks, the only time point for AbT at which areduction in anti-dsDNA titers was observed, median AbT levels were4-5-fold higher compared to those predicted by simulations using PK datafrom the single-dose study in MRL-Fas^(lpr) mice and similar compared tothose predicted by simulation with single-dose data obtained from DBAmice (FIG. 45). This observation is in line with that for the AbS 10mg/kg group. Without intending to be bound by theory, a possibleexplanation for relatively lower levels of AbT at the 2-week time point(compared to those of AbS) and undetectable levels of AbT at later timepoints, is marked MAHA production triggered by multiple administrationsof AbT and the inability of AbT to suppress the MAHA response againstitself.

Example 10.8 PK of AbT in Cynomolgus Monkeys

PK of AbT in female monkeys were determined following a single i.v. (10mg/kg or 100 mg/kg) or s.c. (10 mg/kg) administration. Meanconcentration-time profiles from this study were compared to those forAbS (FIGS. 46 a-b) and mean PK parameters are summarized in Table 18.

The serum concentrations of human anti-IL-21R antibodies were determinedby qualified ELISAs as described in Table 17. The anti-IL-21R ELISA useda monomeric His-tagged IL-21R as a capture reagent and an anti-human-Fcconjugated to HRP as a detector reagent, as described in Example 10.6.TMB was used to produce a colored endproduct to visualize the bound testarticle. The serum concentration of the isotype control antibody inmonkeys (anti-IL-13 antibody) was also measured by ELISA, as describedin Example 10.6. PK parameters were calculated as noted in Example 10.6.

After a single 10 mg/kg intravenous administration of AbT to femalecynomolgus monkeys, the mean serum clearance (CL) was 7.01 ml/hr/kg andthe mean elimination half-life (t_(1/2)) was ˜2.6 days. The meansteady-state volume of distribution (Vd_(SS)) was ˜202 ml/kg and themean exposure (AUC_(0-∞)) of AbT was 1476 μg*hr/ml (Table 18).

After a single 100 mg/kg i.v. administration of AbT to cynomolgusmonkeys, CL was ˜4.87 ml/hr/kg, t_(1/2) was ˜3.7 days, AUC_(0-∞) was˜20955 μg*hr/ml, and Vd_(SS) was ˜146 ml/kg (Table 18). There was nosignificant difference between the mean PK parameters of AbT in monkeysafter a 10 vs. 100 mg/kg single i.v. dose; thus the PK of AbT wereapproximately linear in the 10-100 mg/kg dose range.

After a 10 mg/kg s.c. administration of AbT to monkeys, the mean T_(max)was ˜6 hr and the mean subcutaneous bioavailability was ˜43%. The meant_(1/2) value after a 10 mg/kg s.c. administration to monkeys was 63 hr(˜2.6 days), and similar to that observed after a 10 or 100 mg/kg i.v.administration (Table 18).

TABLE 17 ELISA Summary for AbT in Mouse and Female Monkey Serum Range ofSerum quantitation Minimum used for Species Assay Assay (ng/ml in 100%Required Standard assay strain Capture Detector serum) Dilution curve(ng/ml) diluent Mouse anti-human anti-human 32-500 20 0.78-100 (in S-Drat DBA IgG IgG-biotin 5% serum) Mouse anti-human anti-human 66.8-1260 40 0.514-102 C57BL/6 MRL^(lpr) IgG IgG-biotin (in 2.5% mouse serum)Mouse Monomeric anti-human 45-768 20 1-38.4 (in 5% CD-1 CD-1 His-taggedIgG-HRP serum) mouse Il-21R Monkey Monomeric anti-human 30-512 200.44-38.4 (in Pooled (cyno) His-tagged IgG-HRP 5% serum) cyno Il-21R

TABLE 18 Mean PK Parameters of AbT in Mice and Female Monkeys DoseC_(5 min) or Species (mg/kg), C_(max) ^(a) T_(max) AUC_(0-∞) CL Vd_(ss)t_(1/2) F strain Route (μg/ml) (hr) (μg * hr/ml) (ml/hr/kg) (ml/kg) (hr)(%) Mouse  8, i.p. 21 1 3320 NA NA 80 ND DBA Mouse  10, i.p. 23 6  957NA NA 21 ND MRL^(lpr) Mouse  10, i.v. 70 NA 4551 2.194 572 210  NA CD-1Monkey  10, i.v. 113 ± 14 NA 1476 ± 312 7.01 ± 1.68 202 ± 44 63 ± 24 NAcyno  10, s.c. 11 ± 3 6 ± 0 638 ± 56 NA NA 63 ± 10 43 ± 4 100, i.v. 1850± 415 NA 20955 ± 3702 4.87 ± 0.79 146 ± 24 88 ± 34 NA

TABLE 19 Median AbS and AbT Concentrations After Multiple Dosing toMRL^(lpr) Mice Antibody (Dose) 2 wk 4 wk 6 wk 8 wk 10 wk AbS (2.5 mg/kg)   0*    0* ND* ND ND AbS (10 mg/kg) 137940* 196887* 198707* 68363 49563AbT (20 mg/kg)  94456* 12765   0 0 0

Example 10.9 Pharmacokinetics of ¹²⁵I-D5 in DBA Mice

The ¹²⁵I-anti-murine IL-21R antibody D5-20 (“D5”) was injectedintraperitoneally into nonfasted male DBA mice in a single 8 mg/kg dose.Serum samples were taken at time points from 1-576 hr, and D5 levelswere quantified by measuring trichloroacetic acid (TCA)-precipitableradioactivity. The PK profile of D5 antibody after i.p. dosage to DBAmice was, in general, similar to that of the human anti-IL-21Rantibodies (Table 20, FIG. 47; see also Tables 14 and 18).

TABLE 20 Pharmacookinetic Parameters of ¹²⁵I-D5 in Male DBA Mice C_(max)AUC_(0-∞) t_(1/2) (hr) T_(max) (hr) (μg/mL) (μg * hr/ml) ¹²⁵I-D5 143.9 636 5422

Example 10.10 Pharmacokinetics in Sprague-Dawley Rats After Intravenous,Subcutaneous, or Intraperitoneal Administration of Anti-IL-21RAntibodies

PK of AbS, AbT, AbV, AbU, and AbW, as well as an isotype controlantibody, were examined after a single 10 mg/kg i.v. dose to S-D rats.Bioanalytical assay for quantitation of test article serumconcentrations, an assay for detection of anti-AbS antibodies in ratserum, and pharmacokinetic calculations were performed as described forAbS and AbT for monkeys (Examples 10.6 and 10.8), with the followingmodifications: S-D serum was used as an assay dilluent and LOQ for testarticle serum concentration assay was 45 ng/mL. For the ELISA methodemployed for an isotype control, an anti-human Fc antibody was used asboth the capture and detector.

Following a single 10 mg/kg i.v. dose to S-D rats, antibody AbS waseliminated slowly (CL ˜1.6 mL/hr/kg); albeit significantly faster(p<0.05) than that for an isotype control IgG (˜0.4 mL/hr/kg). (Table 21and FIG. 48 a). All rats (n=6) had a sharp decline in AbS serumconcentration, such that there was no detectable AbS at and after day 15post-dose. This decline in AbS serum concentration was likely related tothe presence of anti-AbS antibodies detected at day 15 and allsubsequent time points in all six animals. It should be noted thatdifferences in concentration-time profiles in rats between the AbS andthe control IgG are not likely to be explained entirely by anti-productresponses, as mean serum concentrations started to diverge as early as24 hr post-dose and differed by more than 4-fold at the one-week timepoint. The concentration-time profile and PK parameters of AbV closelyresembled those of AbS in rats.

After a single 10 mg/kg i.v. dose to rats, AbT elimination was faster(CL ˜2.1 mL/hr/kg) and the resulting AUC_(0-∞) was ˜2-fold lower,compared to AbS. All of six AbT-dosed rats also had a sharp decline inthe serum concentrations in the terminal phase. The concentration-timeprofiles of AbU and AbW and PK parameters (such as AUC_(0-∞),AUC_(0-240hr), and CL) in rats were intermediate between those of AbSand AbT (FIG. 48 b).

TABLE 21 Pharmacokinetic Properties of Anti-IL-21R Antibodies in S-DRats After 10 mg/kg i.v. Administration Compound C_(5mina) AUC_(0-∞)AUC_(0-240h) ^(b) CL Vd_(ss) (n) (μg/ml) (μg * hr/ml) (μg * hr/ml)(ml/hr/kg) (ml/kg) AbS (n = 6) 191 ± 38.5 6361 ± 184 5266 ± 625 1.60 ±0.210 191 ± 33.0 AbT (n = 5) 149 ± 51.1 3233 ± 356 3036 ± 167 3.12 ±0.312 182 ± 46.0 AbU (n = 7) 161 ± 15.0 4371 ± 552 4116 ± 392 2.32 ±0.328 160 ± 24.2 AbV (n = 7) 153 ± 14.4 5541 ± 691 4907 ± 374 1.83 ±0.211 161 ± 20.3 AbW (n = 5) 144 ± 7.78 4002 ± 737 3613 ± 591 2.58 ±0.522 200 ± 36.7 CL = serum clearance. Vd_(ss) = Volume of distributionat steady-state. AUC = Area under the curve. a. Concentration at 5 min,the first sampling time point after IV administration. ^(b)AbS levelswere <LOQ after 240 hr time point in all rats. c. All animals in alldose groups had sharp drop in test article levels during the 168-504 hrperiod. Thus, the terminal phase was not well defined and the resultingapparent t_(1/2) values were driven by the differences in the onset ofthis concentration drop. Therefore, t_(1/2) values were not used forcomparison of PK across constructs and are not shown.

For AbS, PK in rats was also examined after 10 mg/kg s.c. and i.p.administrations, as well as at two dose levels (1 and 10 mg/mg) afteri.v. administration.

At 168-504 hr after a single i.v., s.c., or i.p. administration to rats,there was a sharp decline in AbS levels in all animals (FIG. 49). In thei.v. and s.c. groups, AbS levels were below LOQ for the assay (45.0ng/mL) at 360 hr through the end of the study (840 hr). In the i.p.group, there was no detectable AbS starting at 576 hr through the end ofthe study. Using the paramagnetic bead assay described in Examples 10.4and 11, anti-AbS antibodies were detected in all animals for all dosegroups as early as 360 hr post-dose (the first sampling time-point takenfor anti-AbS antibody evaluation) and persisted through the end of thestudy (840 hr). Log titers for the anti-AbS response reached 4.01to >4.74 units by the end of the study for all samples tested. Thus, thesharp decline in AbS was likely related to the formation of anti-AbSantibodies.

After a single 1 or 10 mg/kg i.v. dose of AbS in rats, the averageexposures (AUC_(0-∞)) were 470±45 or 6361±845 μg·hr/mL, respectively(Table 22). The average steady-state volume of distribution (Vd_(ss))after a single 1 or 10 mg/kg i.v. dose of AbS was low (101±24 or 191±33mL/kg, respectively), suggesting that AbS was mainly distributedthroughout the vascular space and into limited extracellular fluidsafter i.v. administration. The average clearance (CL) of AbS was2.14±0.21 and 1.60±0.21 mL/hr/kg for the 1 mg/kg and 10 mg/kg i.v.doses, respectively. The average elimination half-life (t_(1/2)) was40±10 and 113±24 hr for the 1 mg/kg and 10 mg/kg i.v. doses,respectively. AbS PK parameters were not dose-proportional in the 1-10mg/kg dose range after i.v. administration to rats, as the averagedose-normalized exposures (AUC_(0-∞)/dose), CL, and t_(1/2), weresignificantly different (p<0.01, unpaired t-test) between the 1 and 10mg/kg dose groups.

After a single 10 mg/kg i.p. dose of AbS to rats, the average serumAUC_(0-∞) was 3929±979 μg·hr/mL and the average estimatedbioavailability (BA) was 62±15% (with the AUC_(0-∞) data from the 10mg/kg, i.v. group used for the BA calculation) (Table 22). The averageserum C_(max) was 31±14 μg/mL and was reached at T_(max) of 20±9 hr.After a 10 mg/kg i.p. dose to rats, there was significant interanimalvariability in the apparent terminal t_(1/2) with the average value of78±70 hr.

After a single 10 mg/kg s.c. dose of AbS to rats, the average serumAUC_(0-∞) was 1595±456 μg·hr/mL and the average estimated BA wasrelatively low, 25±7% (with the AUC_(0-∞) data from the 10 mg/kg i.v.group used for the BA calculation) (Table 22). The average serum C_(max)was 8±1 μg/mL and was reached at T_(max) of 77±26 hr. After 10 mg/kgs.c. dose to rats, there was also significant interanimal variability inthe apparent terminal t_(1/2) with the average value of 88±78 hr.

TABLE 22 Mean (±SD) Pharmacokinetic Parameters of AbS in S-D Rats Aftera Single i.v., i.p., or s.c. Dosage Route, C_(5 min or) AUC_(0-∞)/DoseDose C_(max) ^(a) T_(max) AUC_(0-∞) (μg · hr/mL)/ CL t_(1/2) Vd_(ss)BA^(b) Group N (mg/kg) (μg/mL) (hr) (μg · hr/mL) (mg/kg) (mL/hr/kg) (hr)(mL/kg) (%) 1 6  IV, 10 191 ± 38 NA 6361 ± 845 636 ± 85 1.60 ± 0.21 113± 24  191 ± 33 NA 2 6^(e) IV, 1 19 ± 3 NA 470 ± 45  470 ± 45^(e) 2.14 ±0.2^(e )  40 ± 10^(e) 101 ± 24 NA 3 4  IP, 10  31 ± 14 20 ± 9  3929 ±979 393 ± 98 NA 78 ± 70 NA 62 ± 15 4 4^(c) SC, 10  8 ± 1 77 ± 26 1595 ±456 160 ± 46 NA 88 ± 78 NA 25 ± 7  NA = Not applicable. Note: PKparameters were calculated for each individual animal. ^(a)C_(5 min),concentration at the first sampling time point after i.v. administrationis shown for Groups 1 and 2. C_(max) is shown for Groups 3 and 4.^(b)BA, bioavailability after i.p. or s.c. administration, wascalculated using AUC data for Group 1 (10 mg/kg, IV). ^(c)Statisticallysignificant difference from Group 1 (p < 0.01).

The summary of the onset anti-AbS antibody response in all animals,including S-D rats, is summarized in Table 23.

TABLE 23 Onset of Anti-AbS Antibody Response Following a Single Dose ofAbS to Mice, Rats, and Monkeys Dose (mg/kg) % positive at Species,strain Route Onset (hr)^(a) onset Mouse, 8, ip 672  37% DBA Mouse, 10,ip 336 100% MRL-fas^(lpr) 2.5 ip 312 100% Mouse, 10, iv 576  75% CD-1Rat, Sprague- 10, iv 360 100% Dawley Monkey, 10, iv 504 100% Cynomolgus10, sc 504 100%

Example 11 Pharmacokinetics and Biodistribution of ¹²⁵I-AbS in Wild-TypeControl (C57BL/6), IL-21R Knockout, and MRL-Fas^(lpr) Mice Example 11.1¹²⁵I Labeling

Iodination was performed using the IODO-BEADS method (Pierce, Rockford,Ill.) according to the manufacturer's instructions, and purified byfiltration. Briefly, ˜100-200 μg of test article were incubated for 25min with 1-2 mCi of ¹²⁵I (PerkinElmer), three IODO-BEADS, and ˜100-200μl of PBS. The reaction mixture was separated from the IODO-BEADS andtransferred to a Centricon filtration device (10 kD cut-off, Millipore).The purification was performed by adding ˜5-10 ml of PBS (in aliquots of1-2 mL) and spinning at 2,000×g until the volume in the upper chamber ofthe filtration device was down to ˜200-500 μl.

The dosing solution was prepared by combining the stock solution ofunlabeled test article, the formulation buffer, and the ¹²⁵I tracer. Thepurity of the dosing solution was analyzed using reducing andnonreducing SDS-PAGE. The dosing solution was prepared once, one dayprior to dosing of the first cohort of animals.

Example 11.2 Determination of Radioactive Equivalent Concentrations inSerum

Total radioactivity (in counts per min (cpm)) in 50-100 μl of serumsamples (in duplicate) was determined by gamma-counting (1480 WIZARD™,Wallac Inc., Gaithersburg, Md.). An equal volume of 20% TCA(trichloroacetic acid) was added to each aliquot, and samples were spunat 12,000 rpm for 10 min. TCA-soluble radioactivity in the supernatant(with the volume equal to the sample volume used for total radioactivitycount) was determined by gamma-counting. TCA-precipitable radioactivityin a given sample [total cpm—2×TCA—soluble cpm], the specific activityof the dosing solution (cpm/ng), as well as the dates of the sample(t_(S) (day)) and the dosing solution (t_(D) (day)) measurements, wereused to calculate the radioactive equivalent concentration (ng eq/ml) ina given sample using the formula:

[average TCA−precipitable cpm/EXP(−0.693/60.2×(t_(S)−t_(D))]/[specificactivity×sample volume].

Example 11.3 Determination of the Cumulative Total Count (as Percentageof Dose Count) and Free ¹²⁵Iodine Fraction in Urine

Total counts were used to calculate urinary excretion (excreted cpm as %dose) for each collection period, using the formula: [100%×urinecpm/EXP(−0.693/60.2×(t_(S)−t_(D)))]/[specific activity×dose]. Cumulativeradioactivity in the urine (cumulative excreted cpm as % dose) wasdefined as the sum of urinary excretions (cpm, as % dose) from time 0 upto an indicated time point.

To determine the fraction of TCA-soluble radioactivity, 50 μl urinealiquots were mixed with 50 μl of normal mouse serum (resulting in 100μl samples) and analyzed by gamma-counting. A 100 μl aliquot of 20% TCAwas added to each 100 μl sample, and the 200 μl samples were spun at12,000 rpm for 10 min. TCA-soluble radioactivity in the 100 μl ofsupernatant was determined by gamma-counting. The fraction of freeiodine was obtained using the formula: [2×100%×TCA-soluble cpm in 100 μlof supernatant/total cpm in 50 μl of urine].

Example 11.4 Biodistribution Analysis

Tissue samples were placed into preweighed tubes, weighed to determinetissue weights in grams, and counted for total radioactivity (cpm). Thequantitation of radioactive equivalent tissue concentration (ng eq/g)was based on the total radioactivity in tissues and the specificactivity of the dosing solution (cpm/ng) after a correction forhalf-life of ¹²⁵I using the formula: [samplecpm/EXP(−0.693/60.2×(t_(S)−t_(D))]/[specific activity×sample weight].

Tissue to serum concentration ratios (T/S) for a given tissue at a giventime point were calculated using the ratio of radioactive equivalentconcentration in tissue (μg eq/g) to that in serum (μg eq/ml). Totaltissue counts as % dose were calculated using the formula: [100%×samplecpm/EXP(−0.693/60.2×(t_(S)−t_(D))]/[specific activity×dose].

Example 11.5 Pharmacokinetics and Biodistribution in Control and IL-21RKnockout Mice After a 2.5 mg/kg Intravenous Dose

In wild-type C57BL/6 (control) mice, there was a decline in mean AbSserum levels relative to those in IL-21R knockout mice, starting at10-14 days after a single 2.5 mg/kg i.v. dose of ¹²⁵I-AbS (FIG. 50 c).At day 14 (336 hr), 60% of controls (n=10) and 0% of IL-21R knockoutmice (n=10) had undetectable or very low levels of AbS. After day 20(480 hr), there were no detectable AbS levels in the serum of allcontrol mice. In IL-21R knockout mice, AbS was detected in serum as lateas day 36 (864 hr) post-dose, the last sampling time point. Thedifferences in concentration-time profile of ¹²⁵I-AbS in control vs.IL-21R knockout appears to be due to possible differences in anti-AbSresponses in these two mouse strains. At the five-week time point, about55% (five out of nine) wild-type serum samples and none of IL-21R knockout serum samples tested positive in the anti-AbS antibody assay.

In control mice, the total body clearance (CL) was ˜1.3 ml/hr/kg, andthe elimination half-life (t_(1/2)) was 120 hr (˜5.0 days). In IL-21Rknockout mice, CL was ˜0.7 ml/hr/kg, and t_(1/2) was 256 hr (10.7 days)(Table 14). Accordingly, after a 2.5 mg/kg i.v. dose, IL-21R knockoutmice had a higher serum exposure (AUC_(0-∞)) as compared to controls(Table 14).

In general, for both controls and IL-21R knockout mice, the highestconcentrations of ¹²⁵I-AbS were found in serum as compared to othertissues examined (FIGS. 50 a and 50 b; Tables 24 and 25). However, atthe last tissue sampling time point (864 hr), detectable ¹²⁵I-AbS levelswere found in tissues, but not in serum, of control mice.

In accordance with differences in serum levels of AbS between controland IL-21R knockout mice, radioactive equivalent concentrations of AbSin tissues were lower in controls as compared to IL-21R knockouts,starting at ˜2 weeks post-dose (FIG. 50). Likewise, tissue eliminationhalf-lives (t_(1/2)) were shorter in control mice (˜90-165 hr) ascompared to those in tissues of IL-21R knockout mice (˜230-270 hr).Accordingly, the tissue exposures (AUC_(0-∞)) were lower in control micecompared to IL-21R knockout mice (Table 26). Thus, after a 2.5 mg/kgi.v. dose of ¹²⁵I to IL-21R knockout mice, exposure in both serum andtissues was higher as compared to controls.

TABLE 24 Mean (± SD) Tissue to Serum (T/S) Radioactive EquivalentConcentration Ratio After a Single Intravenous Dose of 2.5 mg/kg of¹²⁵I-AbS to C57BL/6 Mice 1 hr 48 hr 168 hr 336 hr fat 0.0099 ± 0.00620.0849 ± 0.0289 0.0619 ± 0.0144 0.2316 ± 0.2619 femurs 0.0360 ± 0.01260.1203 ± 0.0124 0.1108 ± 0.0140 0.6979 ± 1.0550 heart 0.0776 ± 0.01090.1588 ± 0.0143 0.1122 ± 0.0123 0.3837 ± 0.4993 kidney 0.1293 ± 0.03110.2497 ± 0.2105 0.0957 ± 0.0215 0.6350 ± 0.8669 large 0.0122 ± 0.00560.0760 ± 0.0127 0.0466 ± 0.0078 0.2102 ± 0.2544 intestine liver 0.1600 ±0.0770 0.1342 ± 0.0679 0.1206 ± 0.0717 1.1834 ± 1.4455 lungs 0.1968 ±0.0863 0.2522 ± 0.0714 0.1462 ± 0.0776 1.3408 ± 2.4852 lymph 0.0314 ±0.0163 0.1554 ± 0.0333 0.1504 ± 0.0542 3.7491 ± 5.7164 node skeletal0.0099 ± 0.0048 0.0789 ± 0.0108 0.0724 ± 0.0064 0.2461 ± 0.3171 muscleskin 0.0243 ± 0.0195 0.3125 ± 0.0327 0.3145 ± 0.1155 1.4147 ± 1.6123spleen 0.2450 ± 0.0289 0.2043 ± 0.0403 0.1470 ± 0.0232 2.2414 ± 3.4892stomach 0.0471 ± 0.0530 0.0791 ± 0.0249 0.0674 ± 0.014  0.4292 ± 0.5801thymus 0.0215 ± 0.0117 0.0842 ± 0.0174 0.0590 ± 0.0125 0.2794 ± 0.3901Individual values below the limit of quantitation (LOQ, defined as 3Xthe background cpm) were treated as “0” for calculations of the mean andthe standard deviation (SD). T/S were not calculated for time pointsafter 336 hr (480 and 864 hr), as serum concentrations were below thelimit of detection.

TABLE 25 Mean (±SD) Tissue to Serum (T/S) Radioactive EquivalentConcentration Ratio After a Single Intravenous Dose of 2.5 mg/kg of¹²⁵I-AbS to IL-21R Knockout Mice 1 hr 48 hr 168 hr 336 hr fat 0.0063 ±0.0022 0.0718 ± 0.0353 0.0856 ± 0.0475 0.0885 ± 0.0381 femurs 0.0327 ±0.0069 0.1283 ± 0.0555 0.1169 ± 0.0104 0.1238 ± 0.0217 heart 0.0733 ±0.0152 0.1569 ± 0.0172 0.1526 ± 0.0213 0.1681 ± 0.0315 kidney 0.1491 ±0.0417 0.1570 ± 0.0218 0.1588 ± 0.0347 0.1687 ± 0.0417 large 0.0112 ±0.0034 0.0666 ± 0.0133 0.0612 ± 0.0111 0.0568 ± 0.0122 intestine liver0.1550 ± 0.0466 0.1434 ± 0.0706 0.1190 ± 0.0549 0.0784 ± 0.0282 lungs0.3495 ± 0.1671 0.2722 ± 0.0529 0.1178 ± 0.0707 0.2510 ± 0.1016 lymph0.0224 ± 0.0096 0.1337 ± 0.0369 0.1215 ± 0.0345 0.1118 ± 0.0384 nodeskeletal 0.0068 ± 0.0016 0.0663 ± 0.0077 0.0761 ± 0.0115 0.1227 ± 0.1479muscle skin 0.0185 ± 0.0029 0.2716 ± 0.0315 0.3244 ± 0.0607 0.3593 ±0.1247 spleen 0.2682 ± 0.0425 0.1943 ± 0.0304 0.1809 ± 0.0322 0.1846 ±0.0483 stomach 0.0318 ± 0.0151 0.0617 ± 0.0165 0.0689 ± 0.0237 0.0724 ±0.0174 thymus 0.0206 ± 0.0099 0.0929 ± 0.0178 0.0979 ± 0.0127 0.1049 ±0.0233 Individual values below the limit of quantitation (LOQ, definedas 3X the background cpm) were treated as “0” for calculations of themean and the standard deviation (SD). T/S were not calculated for timepoints after 336 hr (480 and 864 hr), as serum concentrations were belowthe limit of detection.

TABLE 26 Exposure of ¹²⁵I-AbS in Tissue of C57BL/6 and IL-21R KnockoutMice Following a Single 2.5 mg/kg Intravenous Dose C57BL/6 IL-21R KOCmax^(a) Tmax AUC_(0-∞) Cmax^(a) Tmax AUC_(0-∞) μg eq/ml hr hr * μgeq/ml μg eq/ml hr hr * μg eq/ml fat 0.66 48 120 0.64 48 236 femurs 0.9348 213 1.17 48 370 heart 1.79 1 258 1.97 1 501 kidnely 3.06 1 352 4.07 1573 large 0.59 48 105 0.61 48 181 intestine liver 3.77 1 317 4.24 1 419lymph node 1.21 48 303 9.88 1 882 lung 4.84 1 447 1.21 48 357 skeletal0.61 48 128 0.60 48 238 muscle skin 2.44 48 567 2.48 48 939 spleen 5.771 454 7.20 1 706 stomach 0.78 1 163 0.81 1 222 thymus 0.65 48 141 0.8548 320 serum 29 NA 1927 32 NA 3415 ^(a)C_(5 min) concentration at thefirst sampling time point is shown for serum. Tissue sampling timepoints were: 1, 48, 168, 336, 480, and 864 hr.

Example 11.6 Pharmacokinetics and Biodistribution in MRL-Fas^(lpr) MiceAfter a 2.5 mg/kg Intraperitoneal Dose

Following a single i.p. dose of 2.5 mg/kg of ¹²⁵I to femaleMRL-Fas^(lpr) mice (˜10-12 weeks old), the serum concentration-timeprofile was biphasic, showing a relatively slower decline in AbS levelsduring the first week post-dose (t_(1/2) ˜54 hr) and a faster declineafter 5 days post-dose (t_(1/2) ˜12 hr), suggesting the formation ofanti-AbS antibodies (FIGS. 51 a and 52). In fact, 4 of 4 mice at the 312hr time point, and 4 of 4 mice at the 408 hr time point, tested positivefor anti-product antibodies (with relative media titers of ˜4.01 and˜4.43 log titer units, respectively). After a 2.5 mg/kg i.p. dose of¹²⁵I, the maximum serum concentration (C_(max)) and exposure (AUC_(0-∞))of AbS were 12 μg eq/ml and 823 μg eq·hr/ml, respectively. Thus, after asingle i.p. dose to MRL-Fas^(lpr) mice, PK were approximately linear inthe 2.5-10 mg/kg dose range (FIG. 52; Table 14) (see also Example 10.4).Urine counts of ¹²⁵I-AbS in MRL-Fas^(lpr) mice were elevated as of the10 day time point (FIG. 51 b).

During the first week following a single 2.5 mg/kg i.p. dose (likelyprior to formation of anti-product antibodies), the highestconcentrations of AbS were found in serum for all tissues examined.After the first week, serum concentration declined more rapidly ascompared to the decline of AbS in tissue, so that T/S concentrationratios were significantly higher than 1 (Table 27), and AUC_(0-∞) intissues was ˜90-300 μg eq·hr/ml (Table 28). In tissue, the apparentelimination half-life (calculated based on the 24-312 hr data set) was˜40-55 hr. Relatively low (but still detectable) levels of radioactivitypersisted in tissues until the end of the study (seven weeks post-dose)(FIG. 51 a).

TABLE 27 Mean (±SD) Tissue to Serum (T/S) Radioactive EquivalentConcentration Ratio After a Single IP Dose of 2.5 mg/kg of ¹²⁵I-AbS toMRL-Fas^(lpr) Mice 24 hr 72 hr 168 hr 240 hr fat 0.321 ± 0.073 0.233 ±0.070 41.125 ± 69.735  71.500 ± 131.496 kidney 0.195 ± 0.026 0.202 ±0.036 9.831 ± 6.787 56.205 ± 25.079 liver 0.104 ± 0.028 0.091 ± 0.0328.067 ± 5.587 16.820 ± 6.892  lungs 0.177 ± 0.074 0.159 ± 0.070 7.621 ±6.512 20.127 ± 12.783 lymph node 0.197 ± 0.055 0.141 ± 0.020 5.490 ±3.707 22.497 ± 16.812 skin 0.318 ± 0.023 0.305 ± 0.056 12.237 ± 8.734 50.869 ± 59.413 spleen 0.166 ± 0.022 0.105 ± 0.020 9.648 ± 6.834 50.418± 33.424 Individual values below the limit of quantitation (LOQ, definedas 3X the background cpm) were treated as “0” for calculations of themean and the standard deviation (SD). T/S were not calculated for timepoints after 240 hr, as serum concentrations were below the limit ofdetection in most of the mice.

TABLE 28 Exposure of ¹²⁵I-AbS in Tissue of MRL-Fas^(lpr) Mice Followinga Single 2.5 mg/kg IP Dose AUC_(0-∞) Cmax Tmax hr * μg μg eq/ml hr eq/mlfat 2.9 24 255 kidney 1.8 24 197 liver 1.0 24 95 lymph node 1.8 24 133lung 1.6 24 150 skin 2.9 24 285 spleen 1.5 24 161 serum 12 3 823 Tissuesampling time points were: 24, 72, 168, 240, 312, 528, 864, and 1176 hr.

Example 12 Inhibitory Properties of Anti-IL-21R Antibodies in HumanBlood Example 12.1 Agonistic Response of Human Whole Blood to IL-21 isNeutralized by Ex Vivo Treatment of Anti-IL-21R Antibody

Human whole blood was drawn by the Human Blood Donor Program inCambridge, Mass. All human blood samples were collected in BDVacutainer™CPT™ cell preparation tubes. Collection tubes containedsodium heparin. Samples were maintained at ambient temperature andprocessed immediately. Blood was divided into 1 to 2 mL aliquots incryovials, treated with IL-21, AbS, or control proteins. When sampleswere treated with both antibody and IL-21, the antibody was addedimmediately prior to IL-21. Samples were then incubated at 37° C. in aForm a Scientific Reach-In Incubator Model #3956 for four hr while mixedcontinuously at 15 RPM using the Appropriate Technical Resources Inc(ATR) Rotamix (Cat. # RKVS) rotating mixer (serial #0995-52 and#0695-36), or using the Labquake® Tube Shaker/Rotator (Cat. #400110)during the incubation. Aliquots (0.5 mL) were removed using a GilsonP1000 pipette with ART 1000E tips (Cat. #72830-042) and added to 2.0 mLmicrotubes (Axygen Scientific, Cat. #10011-744) containing 1.3 mL ofRNAlater® supplied with the Human RiboPure™-Blood Kit (Ambion, Austin,Tex.; Cat. # AM1928) and mixed thoroughly by five complete inversions.Samples were stored at ambient temperature overnight and then frozen at˜80° C. pending RNA purification.

RNA was isolated using the Human RiboPure™-Blood Protocol (Ambion, Cat.# AM1928). The Human RiboPure™ RNA isolation procedure consists of celllysis in a guanidinium-based solution and initial purification of theRNA by phenol/chloroform extraction, and final RNA purification bysolid-phase extraction on a glass-fiber filter. The residual genomic DNAwas removed according to the manufacturer's instructions for DNAsetreatment using the DNA-free™ reagents provided in the kit. For allsamples, RNA quantity was determined by absorbance at 260 nm with aNanoDrop 1000 (NanoDrop, Wilmington, Del.). RNA quality was spot-checkedusing a 2100 Bioanalyzer (Agilent, Palo Alto, Calif.). Samples werestored at −80° C. until cDNA synthesis was performed.

According to the manufacturer's instructions, cDNA was reversetranscribed from total RNA using a High Capacity cDNA ReverseTranscription Kit (ABI, Cat. #4368814) with additional RNase inhibitorat 50 U/sample (ABI, Cat. #N808-0119). cDNA samples were stored at −20°C. until RT-PCR (real-time PCR) was performed. The amount of cDNA loadedon a Taqman® Low Density

Array card was determined using the lowest RNA yield obtained within anexperiment. cDNA samples were assayed on an ABI PRISM 7900 Sequencedetector (Sequence Detector Software v2.2.2, Applied Biosystems) usinguniversal thermal cycling conditions of 50° C. for 2 min, 95° C. for 10min, then 40 cycles of 95° C. for 15 sec and 60° C. for 1 min.

To check for ex vivo effects of IL-21, experiments were conducted totest whether human whole blood responded to IL-21 with detectablechanges in gene expression levels. Whole blood from human donors wasincubated in the presence and absence of IL-21, and RNA levels weredetermined using TLDA cards. Two different TLDAs were used to measureRNA expression levels. The first, Human Immune TLDA (ABI, Catalog#4370573), tested 96 genes, of which 91 were detectable in stimulatedhuman blood. PBMCs stimulated with LPS or PHA from human donor wholeblood was used as a positive control. To test the upregulation of IL-21Rin response to IL-21 stimulation, results were obtained using a customdesigned TLDA that contained the IL-21R gene.

Data for the seven most consistent IL-21-dependent gene expressionchanges observed in whole blood samples of the four humans tested underthe selected assay conditions are shown in Table 29. Significantresponses to IL-21 were observed for IL6, IFNγ, CD19, PRF1, IL10, IL2Rα,and GNLY. Interestingly, IL-21 treatment at concentrations of 30 ng/mL,100 ng/mL and 500 ng/mL produced similar results (Table 29).

TABLE 29 IL-21-dependent gene expression changes in human whole bloodsamples IL21 IL21 IL21 (30 ng/mL) (100 ng/mL) (500 ng/mL) Average FoldAverage Fold Average Fold Composite Composite Change Change ChangeAverage Fold Standard Paired T-test Gene (2 Humans) (4 Humans) (2Humans) Change Deviation (P-Value) IL6 20.17 13.24 32.14 19.70 22.511.35E−01 IFNG 7.65 6.51 7.23 6.97 2.34 5.86E−02 CD19 2.72 2.78 2.78 2.760.94 6.09E−02 PRF1 2.43 2.87 2.72 2.72 1.08 5.21E−02 IL10 3.17 2.49 2.662.70 1.26 3.78E−02 IL2RA 2.82 2.07 2.15 2.28 0.86 5.35E−02 GNLY 2.122.07 2.66 2.23 0.38 1.76E−03 Fold change was calculated by dividing RQof treated group by RQ of control group. Paired T-test values werecalculated using the RQ values and pairing control and treated valuesfrom each human donor.

To determine if AbS had the desired blocking activity ofIL-21/IL-21R-dependent activation of human cells, the ability of thisantibody to block IL-21-dependent activation in whole blood was testedon one of the human donors. FIGS. 53 a-b show the effective inhibition(compared to the Hu IgG1 control) of IL-21-dependent activation ofIL-21-responsive genes by the antibodies.

These results define methods for measuring the response to IL-21 inhuman whole blood samples; thus, this assay can be used to detect theinhibition of IL-21-dependent activation by anti-IL-21R antibodies inexperimental or clinical settings, because the assay does not requiremore blood than can be routinely collected and involves minimal ex vivomanipulation.

Example 12.2 An Assay Measuring PD Activity of AbS in Whole Human Blood

A total of five healthy human donors were used in these studies. Allhuman whole blood samples were collected in BD Vacutainer™ CPT™ cellpreparation tubes containing sodium heparin (Catalog #362753). Humanwhole blood was drawn by the Human Blood Donor Program in Cambridge,Mass. Samples were maintained at ambient temperature and, except whereotherwise stated, were processed within an hour of collection. Whensamples were treated with immunoglobulin reagents and IL-21, theimmunoglobulin reagent was added prior to addition of IL-21. Sampleswere incubated at 37° C. in a Form a Scientific Reach-In Incubator Model#3956 for the duration noted while mixed continuously at about 7 RPMusing the Appropriate Technical Resources Inc (ATR) Rotamix (Catalog#RKVS) rotating mixer (serial #0995-52 and #0695-36), or using theLabquake® Tube Shaker/Rotator (Catalog #400110) during the incubation.Subsequently, 0.5 mL of each sample was removed and added to 2.0 mLmicrotubes (Axygen Scientific, Catalog #10011-744,) containing 1.3 mLsof RNAlater® which is provided with the Human RiboPure™-Blood Kit(Ambion, Catalog # AM1928), and mixed thoroughly by five completeinversions. Following procedure described in Example 12.1, samples wereprocessed to isolate and quantify RNA yield, and synthesize cDNA; and200 ng of cDNA was loaded onto a custom TLDA plate, comprising 24 genes(19 test genes whose RNA expression levels had been observed to changein whole human blood upon stimulation with IL-21, and 5 endogenouscontrol genes), shown in FIG. 54.

For quantification of RNA expression levels, average Real-Time PCRthreshold cycle (Ct) values from several endogenous controls (GAPDH,GUSB, ZNF592, and PGK1 in FIG. 54) were used as “normalizers” becausethe expression of these genes did not change with treatment and thegenes yielded the most consistent Ct values across all samples. AverageCt values of experimental controls (no AbS and no IL-21 added) were usedas “calibrators.” Expression of a gene in a given sample was calculatedas Ct of gene−Ct of average of endogenous controls for that sample (ΔCtof sample). The gene expression value (ΔΔCt) was calculated as ΔCt ofsample−ΔCt of “calibrator.” Relative quantification (RQ) or fold changewas calculated as 2^(−ΔΔCt).

In order to determine optimal time and dose of IL-21 treatment forgeneration of maximal signal, whole blood samples from five healthydonors were incubated in the presence of 3.3, 10 or 30 ng/ml of IL-21for 2, 4, 6 or 24 hr. RNA was isolated and gene expression levelsmeasured. Significant and robust IL-21 dependent signals were obtainedfor six genes: IL6, IFNγ, IL2Rα, GZMB, PRF1, CD19. The optimal signalfor all but CD19 was obtained at 2 hr (FIG. 55). There was littledifference in the response obtained at 3.3, 10 or 30 ng/ml IL-21.Response to ex vivo IL-21 treatment was consistent between all fivedonors (data not shown). Based on the results obtained with these fivedonors, the assay conditions chosen to titrate the inhibitory effect ofAbS on the ex vivo response to IL-21 were: two hr stimulation with 10ng/ml of IL-21. The most reliable IL-21-responsive genes were GZMB,IFNγ, IL-21RA, IL-6, and PRF1.

To determine the dose of AbS to optimally block the effect of IL-21,samples from four individual donors were preincubated for 2 hr at theindicated concentrations of AbS and IgG₁TM, both diluted in PBS, beforethe addition of 10 ng/ml of IL-21. Following the addition of IL-21,samples were incubated for an additional two hr.

Addition of 0.1 μg/mL AbS resulted in full inhibition, so 0.003 μg/mL ofAbS was used for subsequent experiments. AbS, but not IgG1TM, inhibitedthe response of all six genes tested in all four donors, as demonstratedin FIGS. 56 a-c. The IC₅₀ for all six genes is presented in Table 30.

TABLE 30 Inhibitory Activity of AbS on IL-21 Response Signal in WholeBlood CD19 GZMB IFNG IL2Rα IL6 PRF1 IC₅₀ (ug/mL) 0.007 <0.003 0.0150.002 0.005 0.007

As demonstrated in FIG. 56, AbS at concentration as low as 0.03 μg/mL(200 μM or 6×10⁴ molecules/cell) successfully blocked the effects of 10ng/mL of IL-21 on gene expression. This inhibition was reduced when theconcentration of AbS was lowered to <0.003 μg/mL (20 μM). GZMB, IFNγ,IL-2Rα, IL-6, and PRF1 were reliable blood biomarkers of IL-21R/IL-21and antibody activity at 2 hr; however all six genes (GZMB, IFNγ,IL-2Rα, IL-6, PRF1, and CD19) were identified as useful human bloodbiomarkers for IL-21R/IL-21 activity. Moreover, 10 ng/mL of IL-21 wasshown to decrease gene expression of TBX-21 gene in the same assay, andthis decrease was completely reversed by AbS at 0.03 μg/mL (data notshown).

Example 13 PK and PD of AbS and AbT in Male Cynomolgus Monkeys Example13.1 Animal Selection for AbS and AbT Study

Animals used in the study were protein-naïve and selected for inclusionbased on results obtained with recombinant human IL-21 stimulation inthe whole blood ex vivo assay prior to dosing.

Specifically, for male monkeys, whole blood samples (0.5-1.5 mL) wereplaced in sterile, nuclease-free, 2 mL micro-centrifuge tubes (Axygen,cat. #1011-744) and treated with vehicle (10 mM L-histidine, 5%sucrose), 50 ng/mL recombinant human IL-21 (rhuIL-21), 50 ng/mL rhuIL-21with 30 nM IgG control antibody, or 50 ng/mL rhuIL-21 with 30 nManti-IL-21R antibody for 4 hrs at 37° C. on a platform shaker. Forfemale monkeys, whole blood samples (0.5 mL) were treated with eithervehicle or 20 ng/mL rhuIL-21. Peripheral blood mononuclear cells in theblood samples were isolated by Ficoll method according to manufacturer'sinstructions (GE Healthcare, Ficoll-Paque™ plus) and washed once in PBS.

RNA isolation was performed using the RiboPure™-Blood Kit (Ambion,Cat#AM1928; males) or RNeasy kit (Qiagen, females) according tomanufacturer's instructions. RNA yield was determined using a NanoDrop1000A spectrophotometer (NanoDrop, Wilmington, Del.) and RNA quality wasassessed using a 2100 Bioanalyzer (Agilent, Santa Clara, Calif.). RNAconcentration was adjusted to 28 ng/mL (males) or 20 ng/μL (females).

For male monkeys, synthesis of cDNA was performed using a High CapacitycDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif.,cat. #4368814) according to manufacturer's instructions with 700 ng ofRNA and gene expression analysis was performed using a Wyeth custom TLDAcard (Applied Biosystems, part #4342249) designed for detection ofcynomolgus monkey genes. Each cDNA synthesis reaction was mixed withTagMan® 2×PCR Master Mix

(Applied Biosystems, cat. #4304437), and 100 μL was loaded onto a TLDAcard. TLDA cards were processed according to manufacturer instructionsand amplification was performed using an ABI Prism® 7900HT SequenceDetection System. Cycling parameters used for each run were as follows:50° C. for 2 min, 95° C. for 10 min, and 40 cycles of 95° C. for 15 secfollowed by 60° C. for 1 min. Cycle thresholds (C_(T)) were calculatedusing Sequence Detection Software (version 2.3, Applied Biosystems).

For female monkeys, TaqMan quantitative RT-PCR for IL-2Rα only wasperformed using prequalified primers and probes to IL-2Rα (AppliedBiosystems; the same IL-2Rα primers and probes as those in custom TLDAfor male monkeys).

For both males and females, the relative quantification (RQ) of geneexpression was then calculated using the delta delta Ct (ΔΔCt) methodwhere RQ=2^(−ΔΔCt. Zinc finger protein) 592 (ZNF592, males) or proteinkinase G-1 (PKG1, females) was used as the endogenous control and thevehicle control sample was used as the calibrator for RQ calculations.Samples with RQ values greater or equal to 1.5 were considered to havegene expression higher than the corresponding vehicle control sample.

Male monkeys whose blood showed higher expression of several immunefunction-related genes when stimulated ex vivo with IL-21 compared withvehicle (RQ>1.5) were selected for inclusion in this study. The RQvalues of five genes (IL-2Rα, IL-21R, PRF1, GZMB, and IL-6) for ninemonkeys selected for further studies described below, represented byanimal number (Animal #), are shown in Table 31. The group number (A-C)represents whether the animals selected were treated with AbS (Group A),AbT (Group B), or IgG control (Group C) in subsequent experiments.Animals 10-13 were not selected for further studies.

TABLE 31 Relative Quantification (RQ) of Gene Expression Induced by ExVivo Addition of IL-21 to Whole Blood Obtained from Male CynomolgusMonkeys Group; Animal # IL-2Rα IL-21R PRF1 GZMB IL-6 A; 1 2.8 2.5 2.82.0 4.2 A; 2 3.4 2.3 1.2 1.1 1.7 A; 3 5.5 2.9 2.5 1.4 3.6 B; 4 4.1 2.12.5 1.1 4.9 B; 5 5.3 3.2 2.1 2.1 4.1 B; 6 2.8 1.8 1.8 2.0 0.5 C; 7 6.31.9 2.4 1.9 2.6 C; 8 2.2 1.8 1.6 1.6 6.7 C; 9 3.8 2.0 1.9 2.2 1.2 10 2.91.8 0.7 1.0 0.9 11 7.7 3.4 2.6 2.2 2.8 12 4.5 3.6 1.4 1.5 1.1 13 2.1 1.51.1 1.2 1.4

IL-2Rα was determined to have the largest magnitude (highest RQ) andmost consistent change (highest percentage of animals that had RQ>1.5)in IL-21-induced gene expression of the genes evaluated, and wastherefore considered the best single gene for assessing pharmacodynamic(PD) activity of the anti-IL-21R antibodies. Whole blood samples fromall monkeys included in the in vivo study had IL-2Rα RQ values greaterthan 1.5 following ex vivo stimulation with IL-21. However, significantinteranimal variability in IL-2Rα RQ values was observed, with valuesranging from 2.8 to 6.3.

To obtain a larger number of samples for characterization of thedistribution of the IL-2Rα response to rhuIL-21 in the ex vivo assay,blood samples were obtained from 24 additional female cynomolgusmonkeys, stimulated ex vivo with rhuIL-21, and analyzed for IL-2Rα geneexpression by using a quantitative RT-PCR method (IL-21R, PRF1, GZMB,and IL-6 expression was not analyzed for these monkeys). There were nonoticeable differences in IL-2Rα RQ distribution between male and femalemonkeys (with the median±SD RQ values of 3.8±1.7 and 3.0±1.9,respectively) and subsequent analysis of IL-2Rα RQ distribution wasperformed using a combined data set with n=37. All cynomolgus monkeystested had IL-2Rα RQ values greater or equal to 1.5 following ex vivostimulation with rhuIL-21. The median IL-2Rα RQ value (n=37) was 3.2 andthe range was 1.5-8.1.

RQ values for IL-2Rα expression obtained at baseline from 37 monkeyswere log-transformed and the distribution of the RQ and log {RQ} valueswas tested in the Shapiro-Wilk and D'Agostino & Pearson normality tests(GraphPad Prizm 5, GraphPad Software Inc). The normality hypothesis wasrejected for the RQ distribution (p<0.05) but not for log {RQ}distribution (p=0.16 for the Shapiro-Wilk test; and p=0.48 for theD'Agostino & Pearson test). The log-transformed RQ values were fittedinto normal distribution (R²=0.69) using GraphPad Prizm 5 software. Thedistribution of the IL-2Rα RQ values and log transformation (log 2) ofthe RQ values obtained in the ex vivo assay for all 37 monkeys (n=13males; n=24 females) are shown in FIGS. 57 a and 57 b, respectively. Thedistribution of IL-2Rα RQ values appeared approximately log-normal andpassed normality tests. The inclusion criterion for futurepharmacodynamic (PD) studies with anti-IL-21R antibodies in cynomolgusmonkeys was defined as 2.3, based on the formula: log{RQ_(cutpoint)}=mean of the log-transformed RQ values−standard deviationof the log-transformed RQ values. Thus, animals with distribution of theIL-2Rα RQ values greater than 2.3 were considered to be good respondersto IL-21 stimulation. Animals with RQ values of greater than 2.3 (˜81%;30 of 37) were defined as good responders and were considered to satisfythe inclusion criterion for the PD study of anti-IL-21R antibodies.

To confirm that IL-21-induced gene expression was dependent onengagement of cynomolgus monkey IL-21R, monkey whole blood samples wereincubated simultaneously with IL-21 and an anti-IL-21R antibody (AbT; 30nM) prior to RNA isolation and gene expression analysis. As expected, exvivo addition of AbT simultaneously with IL-21 strongly inhibitedIL-21-induced gene expression changes in the whole blood assay (i.e., RQvalue <1.5; FIG. 57 c).

Example 13.2 Study Design for In Vivo Experiments

Nine male protein-naive cynomolgus monkeys that were identified asresponders to IL-21 stimulation in the ex vivo pharmacodynamic (PD)whole blood assay (described in Example 12.1), were dosed with 10 mg/kgof AbS (Group A), AbT (Group B), or IgG control antibody (Group C), withthree animals per group. The dose was administered i.v. (infusion rateof ˜4 mL/min) into the saphenous vein with a dose volume of 2.5 mL/kg.

Blood samples (˜7.0 mL) for the determination of PD activity (all threegroups) were collected into tubes containing sodium citrate as theanticoagulant. Blood samples (˜3.0 mL) for the determination of serumAbS or AbT concentrations and for the evaluation of anti-productantibodies were collected into tubes without anticoagulant, allowed toclot at room temperature for approximately 15 min, and processed forserum collection by centrifugation. The sample collection schedule isshown in Table 32. After day 50, additional sampling time points wereadded for animals 1 and 3 in the AbS group (Group A) to demonstratereversibility of PD activity. Day 1, also referred to as “doseadministration,” is the day on which antibodies were administered to themonkeys. “Pre-dose” refers to sample collection time prior to doseadministration, and “post-dose” refers to sample collection time afterdose administration.

TABLE 32 In Vivo Study Design and Sample Collection in Male CynomolgusMonkeys Group (Dose) Time Sample Animal # (days) collection^(a) A; AbS−13, 1(pre-^(b) and 5 min post-dose), 2, 8, Animals 1-3 (10 mg/kg, IV)15, 22, 36, 50 Animals 1-3 71, 92^(c) Animal 3 92, 106, 113, 134,148^(c) Animal 1 B; AbT −13, 1(pre- and 5 min post-dose), 2, 8, 15,Animals 4-6 (10 mg/kg, IV) 22, 36 Animals 4-6 C; IgG control −13, 1(pre-and 5 min post-dose), 2, 8, 15, Animals 7-9 (10 mg/kg, IV) 22, 36Animals 7-9 ^(a)For Groups A and B, serum was collected to assay fortest article concentrations and anti-product antibodies, and whole bloodwas collected for ex vivo PD assay. For Group C, only whole bloodsamples were collected. ^(b)For animal 1, pre-dose day 1 samples werenot collected. ^(c)Following PD analysis at day 50, additional samplingtime points were included to demonstrate reversibility of PD activity.

Example 13.3 AbS and AbT Serum Concentrations

To determine AbS and AbT serum concentrations, ELISAs described inExample 10 were used. PK studies in cynomolgus monkeys described inExample 10 indicated that following single i.v. administration, AbS wascleared markedly faster compared to AbT. In this study, extensive serumsampling required for determination of a complete set of PK parameterswere not performed because of the relatively large sample volumerequired for the PD assay and limitations on blood volumes that could becollected from each individual cynomolgus monkey. Samples fordetermination of anti-IL-21R serum concentrations were taken only atthose time points at which PD activity was assessed to enablecorrelation between the serum concentrations and PD activity for eachindividual animal. Thus, only elimination half-life (t_(1/2)) wasestimated based on the terminal phases of serum concentration-timeprofiles. The apparent t_(v2) was determined as described in Example 10.

Following a single 10 mg/kg i.v. dose, AbS was eliminated slowly frommale cynomolgus monkeys, with a mean apparent terminal half-life(t_(1/2)) of ˜10.6±3.92 days (Table 33). Up until day 22, AbS serumconcentrations were very similar between all three AbS-dosed animals(FIG. 58). However, at day 36 and later time points, AbS serumconcentrations in animal 2 declined rapidly (to ˜0.6 μg/mL) compared tothose for animals 1 and 3 (to ˜2 μg/mL). At day 50, animal 2 had nodetectable AbS in the serum (less than LOQ of 30 ng/mL), while animals 1and 3 had AbS serum concentrations of ˜0.9-1 μg/mL. Thus, the estimatedt_(1/2) of AbS was shorter for animal 2 (˜6.2 days), compared to thatfor animals 1 and 3 (˜12 and 14 days, respectively).

As expected based on the initial PK studies, after a single 10 mg/kgi.v. dose administration, the serum concentration of AbT declinedmarkedly faster, compared to that of AbS (FIG. 58). All three AbS-dosedmonkeys had similar concentration time-profiles and apparent t_(1/2)values (Table 33), with serum concentrations declining to relatively lowlevels at day 15 (<0.4 μg/mL) and to less than the LOQ at day 22. Theestimated mean t_(1/2) of AbT was 2.3±0.16 days. AbS and AbTconcentrations started to diverge as early as 24 hrs post-dose anddiffered by more than ten-fold at the one-week time point. These dataconfirmed observations from the earlier PK studies in which AbT had ˜5-7fold faster total body clearance (CL), compared to AbS (see, e.g.,Example 10).

TABLE 33 Peak and Last Detectable Concentrations and EliminationHalf-Life After 10 mg/kg IV Administration of AbS and AbT to MaleCynomolgus Monkeys C_(peak) t_(1/2) C_(last) T_(last) Group Animal(μg/mL) (days) (μg/mL) (days) A (AbS) 1 200 12 0.91 50 2 139 6.2 0.56 363 153 14 0.37 71 Mean 164 11 0.61 52 SD 32 3.9 0.27 18 B (AbT) 4 145 2.50.36 15 5 155 2.3 0.26 15 6 201 2.1 0.25 15 Mean 167 2.3 0.29 15 SD 300.16 0.06 0 C_(peak) Concentration at 5 min (also referred to asC_(5min)), the first sampling time point after i.v. administrationt_(1/2) Elimination half-life T_(last) Last time point at which the testarticle concentration was above the lower limit of quantitation (LOQ =30.0 ng/mL) C_(last) Concentration at T_(last)

Example 13.4 AbS and AbT PD Response in Male Cynomolgus Monkeys

Ex vivo whole blood assay for detection of inhibition of IL-21-inducedexpression by anti-IL-21R antibodies was described in Example 12.1. Forall nine monkeys enrolled into this study, each pre-dose and post-dosewhole blood sample was divided into four 1.5 mL aliquots. First andsecond aliquots were treated with either recombinant human IL-21 orvehicle (a calibrator for RQ calculations), and were used to assesswhether the circulating test article affected ex vivo IL-21-inducedIL-2Rα gene expression (i.e., PD activity). The third aliquot wastreated with IL-21 and an anti-IL-21R antibody (30 nM), and the fourthaliquot was treated with IL-21 and an IgG control antibody (negativecontrol for the anti-IL-21R antibody). The third and fourth aliquotswere used to assess whether inhibition of IL-21-induced IL-2Rα geneexpression by the circulating test article in a given post-dose samplewas complete (for time points at which PD activity was observed),whether the return of IL-21-induced gene expression was mediated throughthe IL-21R (for time points at which PD activity was later lost), and tomonitor for the presence of neutralizing anti-product antibodies.

For AbS, full inhibition of IL-21-induced IL-2Rα gene expression (IL-2RαRQ<1.5) was observed immediately after dose administration and persisteduntil at least day 22 for animal 2 and at least day 50 for animals 1 and3, when serum AbS concentrations were at or above 6 nM (0.9 μg/mL) forall three monkeys (FIGS. 59 a-c and Table 33). Ex vivo IL-21-inducedIL-2Rα expression returned to pre-dose values (i.e., PD activity waslost) at day 92 for animals 1 and 3, coincident with the time points atwhich serum concentrations were <LOQ (FIGS. 59 a and c). For animal 2,PD activity was lost at day 36, when serum AbS concentration declined toa relatively low level of ˜4 nM (0.6 μg/mL). For all time pointsexamined in this study, PD activity of AbS appeared all or none, suchthat there was typically either complete inhibition of IL-21-inducedIL-2Rα gene expression (RQ<1.5), or a lack of inhibition (RQ similar tothat in the corresponding pre-dose sample). A partial PD response wasdifficult to differentiate because of the intra-animal variabilityobserved in IL-2Rα RQ values. It is possible that data points withpartial PD responses for AbS would have been observed if additionalsampling time points were collected at the terminal phase. The minimumconcentration that was needed to maintain minimum PD activity of AbS(C_(max)) could not be precisely estimated, but is likely to be ˜4-6 nM.

For AbT, PD activity was also observed immediately after doseadministration and persisted until at least day 8 (RQ<1.5), when serumAbT concentrations were at or above 1.3 μg/mL (FIGS. 60 a-c and Table33). PD activity was lost (RQ>1.5) at day 15 for all three monkeys. Inblood samples obtained at day 15 from animals 5 and 6, IL-2Rα RQ valuesappeared similar to the corresponding pre-dose values (i.e., completeloss of PD activity) and serum AbT concentrations were less than 1.8 nM.There was a partial PD response in blood samples obtained at day 15 fromanimal 4, as the observed IL-2Rα RQ value of 2.7 was less than that atpre-dose (RQ=4.8) and at the subsequent day 22 time-point (RQ=5.3; FIG.60 a). Animal 4 also had a slightly longer estimated t_(1/2) and asomewhat higher AbT serum concentration at day 15 (˜2.5 nM), compared toanimals 5 and 6 (FIGS. 60 a-c). These data suggest that the C_(min) ofAbT needed to maintain PD activity was approximately 2.5 nM.

For the isotype control group, ex vivo-added recombinant human IL-21induced IL-2Rα gene expression in whole blood samples from all threemonkeys at all time points, with noticeable intra-animal variability inthe IL-2Rα RQ values (data not shown).

In agreement with the earlier PK studies (see Example 10), AbT hadfaster elimination in monkeys compared with AbS, with a mean apparentt_(1/2) of 10.6 and 2.3 days for AbS and AbT, respectively. At the day15 time point, PD activity was completely or partially lost in all threeAbT-dosed monkeys, while all three monkeys in the AbS dose group hadrelatively high serum AbS concentrations (˜6.0-7.4 μg/mL) and full PDactivity. Thus, AbS had a longer duration of PD activity and a longert_(1/2) in cynomolgus monkeys.

Example 13.5 Anti-Product Antibody Response

At the first time point where loss of PD activity was observed, ex vivoaddition of an anti-IL-21R antibody simultaneous with rhuIL-21 inhibitedthe induction of IL-2Rα gene expression (RQ<1.5) in all AbS- andAbT-dosed monkeys, indicating that the return of rhuIL-21-induced geneexpression was mediated through the IL-21R and that neutralizinganti-IL-21R antibodies were not present (FIGS. 59 and 60). Ex vivoaddition of AbS continued to demonstrate inhibitory activity atsubsequent time points collected from animals 1 and 3. However, AbS hadno ex vivo inhibitory activity at the day 50 time point from animal 2(FIG. 59). Similarly, AbT had no ex vivo inhibitory activity at days 22and/or 36 collected from all animals in AbT-dosed groups (FIG. 60).These data suggested that animal 2 in the AbS group, and all threeanimals (4-6) in the AbT group, had developed neutralizing anti-productantibodies.

The presence of neutralizing anti-AbT antibodies in AbT-dosed animalswas confirmed using an orthogonal flow cytometric (FACS)-based assay.TF-1 and TF-1/rhuIL-21R (TF-1 cells transfected with rhuIL-21R) weregrown in RPMI media containing 25 ng/ml huGMCSF (R&D Systems). Confluentcell cultures were centrifuged at 300 g for 10 min, resuspended inOptiMEM serum-free medium (Invitrogen Corporation) at 10⁶ cells/mL, andincubated at 37° C. for 2 hr. The cells were then washed in coldPBS/0.5% BSA, resuspended in ice-cold PBS buffer, and kept on ice untilstaining. To determine the EC₅₀ for AbS-biotin and AbT-biotin binding toTF-1/rhuIL-21R cells, both the parental TF-1 and the TF-1/rhuIL-21Rcells (10⁵ cells per test) were incubated with either AbT-biotin, orIgG-biotin control using serial 3-fold dilutions (range=16-0.0002 μg/mL)on ice for 30 min, washed in PBS/0.5% BSA, and then incubated withstreptavidin-allophycocyanin (APC; Invitrogen Corporation). Geometricmean fluorescent intensities (“GMFI”) of the APC channel peaks wascollected on an LSRII flow cytometer (BD Biosciences) and analyzed usingFlowjo 8.3.3 software (Treestar). Linear regression analysis of theplots was performed using Prism 4 for Macintosh v4.0b (GraphPadSoftware, Inc.).

The minimum required dilution (MRD) for testing serum samples in thisassay was determined to be 1:6 in PBS/0.5% BSA. To test for inhibitionof AbT-biotin to TF-1/rhuIL-21R cells (i.e. for the presence ofneutralizing activity), TF-1/rhuIL-21R cells were preincubated with serafrom anti-IL-21R-dosed monkeys (using a 3-fold dilution series startingat the MRD), stained with an anti-IL-21R-biotin (at the estimated EC₅₀concentration), washed in PBS/0.5% BSA, stained with streptavidin-APC,and analyzed for GMFI as described above. Each serum sample was run induplicate in two individual experiments, and the average GMFI value forthe four replicates was obtained for each dilution point. The relativeGMFI value for each serum sample for each dilution point was calculatedusing the formula [100%* average GMFI/average GMFI pre-dose]. A samplewas considered positive if the relative GMFI value was less than orequal to 80% at the MRD. For positive samples, the log titer wascalculated as the log [reciprocal dilution that would generate relativeGMFI >80%]. Based on the MRD, log titers for negative samples werereported as <0.78 (log 6).

All three AbT-dosed animals tested positive in the FACS-basedneutralizing antibody assay at days 22 and 36, with log titers rangingfrom 2.2 to 4.1 (Table 34).

TABLE 34 Formation of Neutralizing anti-AbT Antibodies (log Titer) After10 mg/kg i.v. Administration of AbT to Male Cynomolgus Monkeys TIME(DAYS) ANIMAL 4 ANIMAL 5 ANIMAL 6 Pre-dose Negative Negative Negative 15Negative Negative Negative 22 4.12 2.7 2.2 36 2.2  2.7 2.7

As only one of the AbS-dosed animals showed evidence of neutralizinganti-AbS antibodies in the ex vivo IL-2Rα gene expression assay, serumsamples from AbS-dosed monkeys were tested in an electrochemiluminescentparamagnetic bead-based assay described in Example 11.5 that detectedboth neutralizing and normeutralizing anti-AbS antibodies. In thisassay, serum samples were coincubated with biotinylated-AbS andruthenylated-AbS, streptavidin-coated paramagnetic beads were added tothe mixture, and the emitted light was detected using BioVeristechnology. All three AbS-dosed monkeys were positive for anti-AbSantibodies in this assay, with log titers ranging from 1.86 to 3.43(Table 35). There was significant interanimal variability in theapparent onset of anti-AbS generation. The first serum sample that waspositive for anti-AbS antibodies in the BioVeris-based assay wasobtained at days 134, 36, and 92 for animals 1, 2, and 3, respectively.Thus, among the three AbS-dosed animals, animal 2 had the shortestt_(1/2) and the fastest onset and highest titer of anti-AbS antibodyresponse. Animal 2 was also the only AbS-dosed monkey that showedevidence of neutralizing anti-AbS antibody response in the ex vivoIL-2Rα gene expression assay, similar to all three AbS-dosed monkeys.

TABLE 35 Formation of Anti AbS Antibodies (log Titer) After 10 mg/kgi.v. Administration of AbS to Male Cynomolgus Monkeys TIME (DAYS) ANIMAL1 ANIMAL 2 ANIMAL 3 Pre-dose Negative Negative Negative 15 NegativeNegative Negative 22 Negative Negative Negative 36 Negative 2.13Negative 50 Negative 3.43 Negative 71 ND ND Negative 92 Negative ND 2.27106  ND ND 2.79 113  Negative ND ND 134  1.86 ND ND 148  2.4  ND ND “ND”= Not determined

Example 14 PK and PD of Additional Anti-IL-21R Antibodies in CynomolgusMonkeys

Pharmacokinetics of AbV, AbU, and AbW were examined after a single i.v.dose (10μ, 10, and 1 mg/kg, respectively) to protein-naïve cynomolgusmonkeys, and PK parameters were compared to earlier PK data obtainedwith AbS and AbT (Table 36 and FIG. 61). Bioanalytical assays and PKcalculations were performed as described for AbS and AbT in Example 13.

For all compounds, there was significant interanimal variability in theterminal phase, likely related to different onset of formation ofanti-product antibodies. Following a single 10 mg/kg i.v. dose tomonkeys, AbV and AbS appeared to have similar mean serum concentrationsand PK parameters. After a single i.v. dose to monkeys, AbV and AbSappeared to have the slowest mean CL, longest mean t_(1/2), and highest(dose-normalized) mean serum concentrations among all human anti-IL-21Rantibodies tested. AbT had the fastest mean CL, shortest mean t_(1/2)and lowest (dose-normalized) mean serum concentrations among all humananti-IL-21R antibodies tested. Comparison of mean concentration profilesand PK parameters after a single i.v. dose to cynomolgus monkeyssuggested that ranking of human anti-IL-21R Ab antibodies based on PKprofiles was similar between the S-D rats and cynomolgus monkeys (seealso Example 10.10).

TABLE 36 PK Parameters After a Single i.v. Administration of HumanAnti-IL-21R Antibodies to Cynomolgus Monkeys AUC_(0-∞)/ Dose DoseC_(5 min) ^(a) AUC_(0-∞) (μg * hr/ml)/ CL Vd_(ss) t_(1/2) Compound Sex(mg/kg) (μg * hr/ml) (μg * hr/ml) (mg/kg) (ml/hr/kg) (ml/kg) (hr) AbS FMEAN  2 33.3 1575 788 1.28 122 78.5 SD 3.84 184 91.9 0.150 25.0 16.4 FMEAN 10 177 9728 973 1.03 125 74 SD 10 621 62.1 0.07 16 46 M MEAN 10^(b) 164 ND ND ND ND 255 SD 32 ND ND ND ND 94 M MEAN 100  2030 92867929 1.08 211 279 SD 95 9768 97.7 0.108 8.00 27.6 AbT F MEAN 10 113 1476148 7.01 202 63 SD 14 312 31.2 1.68 44 24 M MEAN  10^(b) 167 ND ND ND 55SD 30 ND ND ND 4 M MEAN 100  1850 20955 210 4.87 146 87.8 SD 415 370237.0 0.790 24.4 34.0 AbW F MEAN  1 10.0 430 430 2.33 165 73.2 SD NA NANA NA NA NA NA AbV F MEAN 10 152 9895 989 1.00 228 201 SD 46.0 3181 3180.367 70.4 113 AbU F MEAN 10 197 6807 1947 1.55 140 89.6 SD 35.5 681 1950.447 25.6 35.2 All studies were run in protein-naive monkeys, n = 3 perdose group, unless otherwise noted ^(a)C_(5 min) = concentration at 5min, the first time point after IV administration ^(b)PK data from aseparate PK-PD study NA = Not applicable (n = 2) ND = Not determined;insufficient sampling time points

In the same study, PD activity of AbU-AbW (AbU, AbV, and AbW) in monkeyswas examined using ex vivo rhuIL-21-induced gene expression assay(described in Example 13).

At five min, the first sampling time point, AbU-AbW had PD activity inall monkeys, i.e., displayed complete inhibition of rhuIL-21-inducedgene expression in the ex vivo whole blood assay, similar to dataobtained for AbS and AbT. PD activity was lost with the washout ofAbU-AbW from serum.

Example 15 Pharmacokinetics of AbS After Intravenous Administration toTetanus-Toxoid-Challenged Male and Female Cynomolgus Monkeys

PK of AbS was examined after a single three weekly i.v. administrationof 2 or 10 mg/kg of AbS to tetanus-toxoid-challenged cynomolgus monkeys.AbS serum concentrations and anti-AbS antibodies were monitored byspecific ELISA, and PK parameters were calculated by noncompartmentalanalysis, as described, e.g., in Example 13.

Following three weekly i.v. administrations of 2 or 10 mg/kg to monkeys,AbS concentration-time profiles and PK parameters were generally similarbetween the male and female monkeys in the same dose group (n=3 per sexper group). The mean concentration at 5 min after the 1^(st) dose(C_(5min)) and the 3^(rd) dose (C_(day14, 5min)), as well as the meanexposure after the 3^(rd) dose (AUC_(day14-day21)) increased with thedose level. In the 2 mg/kg group, the mean C_(5min) was 28.4±2.50 μg/mL,the mean C_(day14, 5min) was 35.7±11.0 μg/mL, and the meanAUC_(day14-day21) was 1279±592 μg·hr/mL. In the 10 mg/kg group, the meanC_(5min) was 120±59.9, the mean C_(day14, 5min) was 152±21.9 μg/mL, andthe mean AUC_(day14-day21) was 7700±782 μg·hr/mL. Elimination half-life(t_(1/2)) after the third IV administration of AbS was 25.6±22.7 and168±56.5 hr in the 2 and 10 mg/kg groups, respectively (p=0.0002) (FIG.62).

In the 2 mg/kg group, AbS concentrations declined rapidly after thethird i.v. administration, and all six monkeys tested positive foranti-AbS antibodies at day 28 (two weeks after the third dose) throughthe end of the study, in line with the relatively short t_(1/2) (FIG. 62a). In the 10 mg/kg group, two of the six monkeys (SAN 7 and SAN 8)tested positive for anti-AbS antibodies (at day 42 or 49), and thesemonkeys had the shortest t_(1/2) values in the dose group, suggestingthat formation of anti-AbS antibodies correlated with the t_(1/2) of AbS(FIG. 62 b).

Example 16 Prediction of Pharmacokinetics of AbS and AbT in Humans AfterSingle Administration

To predict the PK of AbS and AbT in humans, two approaches were used.For the first approach, it was assumed that the PK parameters (such asCL and volume of distribution) of an anti-IL-21R Ab in humans would besimilar to those in cynomolgus monkeys. For the second approach,allometric scaling was used to estimate human PK parameters based on theanimal data obtained from mice, rats, and cynomolgus monkeys followingsingle i.v. administration of an anti-IL-21R antibody. Allometricscaling of CL was performed using adjustment for maximal life-spanpotential (MLP) for AbS and brain weight adjustments for AbT, accordingto the methods previously described in Mahmood (1996) Eur. J. DrugMetab. Pharmacokinet. 21:275-78; Mahmood and Balian (1996) Xenobiotica26:887-95; Sacher, “Relation of lifespan to brain weight and body weightin mammals” in CIBA Foundation Symposium—The Lifespan of Animals(Colloquia on Aging), 115-33 (Wolstenholme GEW, O'Connor M, eds, 1959);and Hahn M E, Haber S B. (1978) Behav Genet., 8:251-260. The allometricscaling data for AbS and AbT is demonstrated in FIGS. 63 a-b and 64a-b,respectively.

Collectively, these two approaches suggested a low clearance (CL) and asmall steady-state volume of distribution (Vd_(SS)) of AbS in humans.The predicted CL of AbS would be ˜0.72-1.3 mL/hr/kg and the estimatedVd_(SS) would be ˜92.4-125 mL/kg. Following an i.v. dose of 1 mg/kg ofAbS to a 60 kg human subject, the estimate of exposure (AUC_(0-∞)) is1393 μg·hr/mL, based on the CL value obtained by the allometric scalingmethod. For AbT, these two approaches suggested a higher CL of ˜5-8.5mL/hr/kg and Vd_(SS) of ˜115-202 mL/hr/kg.

Example 17 Treatment with AbS does not Affect Disease in the NZBWF1/JMurine Lupus Nephritis Model

The anti-IL-21R antibody AbS was tested for its ability to reducedisease in a murine model of lupus, using NZBWF/1J mice. Female NZBWF1/Jmice spontaneously develop symptoms resembling those observed in humanlupus nephritis, including high titers of circulating IgG anti-nuclearand anti-double-stranded DNA autoantibodies, IgG deposits in theglomeruli, and proteinuria. The onset of kidney disease, as measured bythe presence of protein in the urine, occurs at approximately 26 weeksof age in female NZBWF1/J mice. To examine the effects of IL-21Rblockade with AbS, 26 week old female NZBWF1/J mice were administeredeither saline (vehicle control), CTLA-4Ig (murine IgG2a, positivecontrol), anti-E.tenalla antibody (murine IgG2a isotype control), AbS,or a human antibody isotype control (human IgG1 antibody with triplemutation) at a dosage of 400 μg/mouse 3×/week over 10 weeks via i.p.injection. Serum samples were taken every two weeks and assayed for IgGanti-dsDNA antibodies by ELISA. Urine was collected and examined everytwo weeks for protein levels using Albustix (Bayer HealthCare,Tarrytown, N.Y.). All groups of animals had similar levels ofproteinuria at the onset of the study, and the degree of proteinuriaescalated in the saline, anti-E.tenella, and hIgG1TM control over thecourse of the study (FIG. 65 a). Treatment with CTLA-4Ig protein, whichhas previously been shown to ameliorate disease in this model, preventedthe development of increased proteinuria in these mice. In contrast,treatment with AbS did not affect the development of proteinuria inNZBWF1/J mice when compared to control mice (FIG. 65 a). Similarly,treatment with CTLA-41 g significantly reduced anti-dsDNA IgG serumantibody titers in NZBWF1/J mice, where treatment with AbS did notaffect the development of anti-dsDNA antibodies in these mice (FIG. 65b).

Example 18 Effects of Treatment with Anti-IL-21R Antibodies in aSemi-Therapeutic Collagen-Induced Arthritis (CIA) Mouse Model

Antibodies AbS and AbT were tested for their ability to reduce diseasein a murine collagen-induced arthritis model of rheumatoid arthritis.Female DBA/1 mice were immunized intradermally in the tail with 100 mgbovine collagen type II emulsified in complete Freund's adjuvant, andthen boosted with 100 mg bovine collagen type II emulsified inincomplete Freund's adjuvant 21 days later at the same site. After thesecond immunization with bovne collagen type II, paws were examined forswelling and scored for severity on a scale of 0-16, with 0 representingno swelling and 16 indicating severe disease. Once 10% of animals in thestudy showed signs of disease, animals were either left untreated, ordosed 3×/week for 30 days with 8 mg/kg either murine IgG2a isotypecontrol antibody, anti-mouse IL-21R antibody D5 (murine IgG2a antibody),mTNFRII-Fc (positive control, murine IgG2a isotype), anti-IL-13™antibody (human IgG1 isotype control antibody), AbT, or AbS. Overalldisease severity in this study was not observed to be as severe asnormally observed in this model. Mean disease severity in the untreatedand isotype control treated mice on the final day of the study was lessthan 4, on a scale of 0-16 (FIG. 66 c). Additionally, although treatmentwith the mTNFRII-Fc positive control reduced disease in this study,statistically significant differences in disease severity were onlyobserved between this treatment group and isotype control treated groupdisease on day 22 after dosing. Treatment with anti-mouse IL-21Rantibody (D5) or anti-human IL-21R antibodies (AbS and AbT) also did notaffect disease severity in this study (FIGS. 66 a-b).

Example 19 Testing Antibody Effects on the Formation of AmnesticAntibody Response to Tetanus Immunization in Cynomolgus Monkeys

The anti-IL-21R antibody AbS was examined for its effects on thedevelopment of amnestic antibody responses to tetanus immunization incynomolgus monkeys. Nine female and nine male cynomolgus monkeys weretested for serum antibody titers to tetanus toxoid to identifytetanus-naïve animals. These animals were then administered 0.5 ml oftetanus toxoid in two equally divided (0.25 ml) doses intramuscularly,and blood was collected every 7 days for 35 days and examined foranti-tetanus IgM and IgG serum antibodies by ELISA. Forty-three daysafter primary immunization, groups of 3 male and 3 female cynomolgusmonkeys were randomly assigned and administered either saline (vehicle),2 mg/kg AbS, or mg/kg AbS as an i.v slow bolus into thebrachial/cephalic or saphenous vein at a dose volume of 1 ml/kg (flushedwith 2 ml vehicle) 1×/week for 3 weeks. Twenty-four hr afteradministering the first dose of AbS or vehicle, monkeys were immunized asecond time with 0.5 ml of tetanus toxoid in two equally divided (0.25ml) doses intramuscularly, and blood was collected routinely andexamined for anti-tetanus IgM and IgG antibody titers by ELISA. IgM andIgG tetanus serum antibodies were detectable in both male and femalecynomolgus monkeys within 14 days after the first immunization withtetanus toxoid (FIG. 67 a). Tetanus specific serum IgM titers did notchange following secondary immunization with tetanus toxoid in any ofthe treatment groups. Tetanus-specific IgG serum titers wereapproximately 10-20 fold greater, and generated more rapidly in salinetreated animals following secondary immunization, consistent with thekinetics of amnestic antibody responses (FIG. 67 b). Treatment with 2mg/kg or 10 mg/kg AbS did not affect the development of tetanus-specificIgG serum antibody responses in cymolgus monkeys following secondaryimmunization, indicating that treatment with AbS does not affect theformation of amnestic antibody responses to tetanus toxoid using thistreatment protocol (FIG. 67 b).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating or preventing an IL-21R-associated disorder in asubject, comprising administering to the subject a binding protein orantigen-binding fragment thereof that specifically binds to human IL-21Rin an amount sufficient to inhibit or reduce immune cell activity in thesubject thereby treating or preventing the disorder, wherein the bindingprotein or antigen-binding fragment thereof comprises at least one aminoacid sequence that is at least about 95% identical to an amino acidsequence(s) selected from the group consisting of: (a) SEQ ID NO:163;(b) SEQ ID NO:164; (c) SEQ ID NO:169; (d) SEQ ID NO:194; (e) SEQ IDNO:195; (f) SEQ ID NO:176; (g) SEQ ID NO:219; (h) SEQ ID NO:219 fromamino acid 1 to 118; (i) SEQ ID NO:220; and (j) SEQ ID NO:221.
 2. Themethod of claim 1, wherein the binding protein or antigen-bindingfragment is an antibody.
 3. The method of claim 1, wherein the bindingprotein or antigen-binding fragment is an scFv. 4-6. (canceled)
 7. Amethod of treating or preventing an IL-21R-associated disorder in asubject, comprising administering to the subject a binding protein orantigen-binding fragment thereof that specifically binds to human IL-21Rin an amount sufficient to inhibit or reduce immune cell activity in thesubject thereby treating or preventing the disorder, wherein the bindingprotein or antigen-binding fragment thereof comprises at least one aminoacid sequence encoded by a nucleotide sequence that is at least about95% identical to a nucleotide sequence(s) selected from the groupconsisting of: (a) SEQ ID NO:239 from nucleotide 148 to 165; (b) SEQ IDNO:239 from nucleotide 208 to 255; (c) SEQ ID NO:239 from nucleotide 352to 378; (d) SEQ ID NO:97 from nucleotide 124 to 156; (e) SEQ ID NO:97from nucleotide 202 to 222; (f) SEQ ID NO:97 from nucleotide 319 to 354;(g) SEQ ID NO:239 from nucleotide 58 to 1401; (h) SEQ ID NO:239 fromnucleotide 58 to 411; (i) SEQ ID NO:97 from nucleotide 58 to 702; and(j) SEQ ID NO:97 from nucleotide 58 to
 384. 8-12. (canceled)
 13. Amethod of treating or preventing an IL-21R-associated disorder in asubject, comprising administering to the subject a binding protein orantigen-binding fragment thereof that specifically binds to IL-21R,wherein the binding protein or antigen-binding fragment thereofcomprises a light chain and a heavy chain, and wherein the light chaincomprises at least one amino acid sequence selected from the groupconsisting of: (a) SEQ ID NO:194; (b) SEQ ID NO:195; (c) SEQ ID NO:176;(d) SEQ ID NO:220; and (e) SEQ ID NO:221. 14-16. (canceled)
 17. A methodof treating or preventing an IL-21R-associated disorder in a subject,comprising administering to the subject a binding protein orantigen-binding fragment thereof that specifically binds to a humanIL-21R epitope that is recognized by AbS, wherein the binding protein orantigen-binding fragment competitively inhibits the binding of AbS tohuman IL-21R, in an amount sufficient to inhibit or reduce immune cellactivity in the subject thereby treating or preventing the disorder.18-20. (canceled)
 21. The method of claim 1, wherein theIL-21R-associated disorder is selected from the group consisting ofautoimmune disorders, inflammatory conditions, allergies, transplantrejections, and hyperproliferative disorders of the blood.
 22. Themethod of claim 21, wherein the IL-21R-associated disorder is selectedfrom the group consisting of multiple sclerosis, systemic lupuserythematosus, psoriasis, transplant rejection, rheumatoid arthritis,and other arthritic disorders.
 23. The method of claim 1, wherein thebinding protein or antigen-binding fragment thereof has an associationconstant for human IL-21R of at least 10⁵ M⁻¹s⁻¹.
 24. The method ofclaim 1, wherein the binding protein or antigen-binding fragment thereofinhibits IL-21-mediated BAF3 cell proliferation with an IC₅₀ of about1.75 nM or less, and wherein the BAF3 cells comprise a human IL-21receptor.
 25. The method of claim 1, wherein the binding protein orantigen-binding fragment thereof inhibits IL-21-mediated proliferationof TF1 cells with an IC₅₀ of about 14 nM or less, and wherein the TF1cells comprise a human IL-21 receptor.
 26. The method of claim 1,wherein the binding protein or antigen-binding fragment thereof inhibitsIL-21-mediated proliferation of primary human B cells with an IC₅₀ ofabout 1.9 nM or less, and wherein the B cells comprise a human IL-21R.27. The method of claim 1, wherein the binding protein orantigen-binding fragment thereof inhibits IL-21-mediated proliferationof primary human CD4⁺ cells with an IC₅₀ of about 1.5 nM or less, andwherein the CD4⁺ cells comprise a human IL-21R.
 28. A method ofdetermining whether an anti-IL-21R antibody is a therapeutic anti-IL-21Rantibody comprising the steps of: (a) contacting a first blood samplefrom a subject with an IL-21 ligand; (b) determining a level ofexpression of at least one IL-21-responsive gene in the first bloodsample contacted with the IL-21 ligand; (c) contacting a second bloodsample from the subject with the IL-21 ligand in the presence of ananti-IL-21R antibody; (d) determining the level of expression of the atleast one IL-21-responsive gene in the second blood sample contactedwith the IL-21 ligand in the presence of the anti-IL-21R antibody; and(e) comparing the levels of expression of the at least oneIL-21-responsive gene determined in steps (b) and (d), wherein a changein the level of expression of the at least one IL-21-responsive geneindicates that the anti-IL-21R antibody is a therapeutic antibody.29-30. (canceled)
 31. A method of determining the pharmacodynamicactivity of an anti-IL-21R antibody comprising detecting a modulation ina level of expression of at least one IL-21-responsive gene in a bloodsample of a subject. 32-35. (canceled)